Seed shattering

ABSTRACT

A plant is provided which contains at least one dehiscence zone (DZ)-selective chimeric gene incorporated in the nuclear genome of its cells, wherein said DZ-selective chimeric gene comprises the following operably linked DNA fragments:  
     a) a transcribed DNA region encoding:  
     1) a RNA which, when produced in cells of a particular DZ of the plant, prevents, inhibits or reduces the expression in said DZ cells of an endogenous gene of the plant encoding a cell wall hydrolase, particularly an endopolygalacturonase, or,  
     2) a protein or polypeptide, which when produced in said DZ cells, kills or disables them or interferes with their normal metabolism, physiology or development,  
     b) a plant expressible promoter which directs expression of said transcribed DNA region at least in said DZ cells, provided that if said transcribed DNA region encodes a protein or polypeptide, or encodes an antisense RNA or ribozyme directed to a sense RNA encoded by an endogenous gene that is expressed in said plant in cells other than said DZ cells, said plant expressible promoter is a DZ-selective promoter, which directs expression of said transcribed region selectively in said DZ cells and wherein said plant is characterized by modified dehiscence properties, preferably delayed dehiscence, when compared to a plant not containing said DZ-selective chimeric gene.

[0001] The present invention relates to DNA sequences, comprisingnucleic acid fragments encoding dehiscence zone-selective proteins,particularly cell wall hydrolases such as polygalacturonases, theregulatory regions of the corresponding plant genes and their use formodifying dehiscence properties in plants, more particularly poddehiscence properties in Brassica napus.

BACKGROUND OF THE INVENTION

[0002] Loss of yield due to seed shedding by mature fruits or pods, alsocalled pod dehiscence or pod shatter, as well as concomitant increase involunteer growth in the subsequent crop year, are a universal problemwith crops that develop dry dehiscent fruits. An economically importantcrop to which these adverse properties specifically apply is oilseedrape: up to 50% of the potential yield may be lost under adverse weatherconditions.

[0003] Dry dehiscent fruits, also commonly called pods, may develop froma single carpel (such as the legume in many Fabaceae) or from more thanone carpel (such as the silique in many Brassicaceae). In case of thesilique, the pod consists of two carpels joined margin to margin. Thesuture between the margins forms a thick rib, called replum. As podmaturity approaches, the two valves separate progressively from thereplum, eventually resulting in the shattering of the seeds that wereattached to the replum.

[0004] Ultrastructural investigation have demonstrated that pod shatteris associated with the precise degradation of cell wall material at thesite of pod valve separation (i.e., the suture). The degradation of thecell wall and loss of cellular cohesion prior to dehiscence ispredominantly attributed to solubilization of the middle lamella of thecell wall. This middle lamella is found between primary cell walls andis the cement that holds the individual cells together to form a tissue.Cell separation is preceded by an ethylene climacteric, which temporallycorrelates with a tissue-specific increase in the activity of thehydrolytic enzyme cellulase (beta-1,4-glucanase) and this occursspecifically in a layer of cells along the suture, which is called thedehiscence zone. In contrast, the activity of the cell wall degradingenzyme polygalacturonase exhibits no correlation either temporally orspatially with pod dehiscence [Meakin and Roberts (1990), J. Exp. Bot.41; 1003]. Pod dehiscence at an early stage of development ischaracteristic of infestation by the pod midge Dasineura brassicae. Alocalized enhancement of both polygalacturonase and cellulase activityhas been observed. However, regulation of midge-induced andmaturation-associated shatter was found to be different [Meakin andRoberts (1991), Annals of Botany 67: 193].

[0005] At first sight, the process of pod dehiscence shares a number offeatures with abscission wherein plants shed organs, such as leaves,flowers and fruits. It has been observed that ethylene induces oraccelerates abscission, whereas auxin inhibits or delays abscission. Adecisive step in abscission is the highly coordinated expression,synthesis and secretion of cell wall hydrolytic enzymes in a discretelayer of cells, called the abscission zone. Cellulases(beta-1,4-glucanases) constitute one class of such cell wall hydrolases.Cellulase activity has been identified in various tissues including leafabscission zones, fruit abscission zones, ripening fruit, senescentcotyledons and styles and anthers [Kemmerer and Tucker (1994), PlantPhysiol. 104: 557 and references therein]. A second class of hydrolasesinvolved in abscission of mainly fruits are polygalacturonases of whichdistinctive isoforms have been identified [Bonghi et al.(1992), PlantMol. Biol. 20: 839; Taylor et al (1990) Planta 183: 133].

[0006] Kadkol et al [(1986), Aust. J. Biol. 34: 79] reported increasedresistance towards shattering in a single, Australian accession of rape.Variation in pod maturation has further been observed in mutants of rapestemming from irradiated seeds [Luczkiewicz (1987), Proc. 7th Int.Rapeseed Congress 2: 463]. It can however be concluded that traditionalmethods for breeding have been unsuccessful in introducing shatterresistance into rape cultivars, without interference in other desirabletraits such as early flowering, maturity and blackleg resistance[Prakash and Chopra (1990), Genetical Research 56: 1].

[0007] Despite its economic impact very little is known concerning themolecular events and changes in gene expression that occur duringoilseed pod dehiscence. At present, two pod-specific mRNAs whoseexpression is spatially and temporally correlated with pod developmenthave been described. However, the function of the encoded proteins isunknown. [Coupe et al (1993), Plant Mol. Biol. 23: 1223; Coupe et al.(1994), Plant Mol. Biol. 24: 223]. PCT publication WO94/23043 in generalterms describes an approach for regulating plant abscission anddehiscence.

[0008] Accordingly, it is an object of the present invention to providedehiscence zone-selective genes in plants.

[0009] These and other objects are achieved by the present invention, asevidenced by the summary of the invention, description of the preferredembodiments and claims.

SUMMARY OF THE INVENTION

[0010] The present invention provides dehiscence zone(“DZ”)-selectivegenes of plants, cDNAs prepared from mRNAs encoded by such genes, andpromoters of such genes. In particular this invention provides the cDNAof SEQ ID NO: 1 and the promoter of a gene encoding a mRNA wherein acDNA of that mRNA has substantially the nucleotide sequence of SEQ IDNO: 1, particularly the promoter as contained within the 5′ regulatoryregion of SEQ ID NO: 14 starting at position 1 and ending at position2,328.

[0011] In another aspect, the present invention also providesDZ-selective chimeric genes, that can be used for the transformation ofa plant to obtain a transgenic plant that has modified dehiscenceproperties, particularly modified pod-dehiscence properties, whencompared to plants that do not contain the DZ-selective chimeric gene,due to the expression of the DZ-selective chimeric gene in thetransgenic plant.

[0012] In yet another aspect, the present invention thus provides aplant containing at least one DZ-selective chimeric gene incorporated inthe nuclear genome of its cells, wherein said DZ-selective chimeric genecomprises the following operably linked DNA fragments:

[0013] a) a transcribed DNA region encoding:

[0014] 1) a RNA which, when produced in the cells of a particular DZ ofthe plant, prevents, inhibits or reduces the expression in such cells ofan endogenous gene of the plant, preferably an endogenous DZ-selectivegene, encoding a cell wall hydrolase, particularly anendo-polygalacturonase (an “endo-PG”), or,

[0015] 2) a protein or polypeptide, which when produced in cells of theDZ, kills or disables them or interferes with their normal metabolism,physiology or development,

[0016] b) a plant expressible promoter which directs expression of thetranscribed DNA region at least in cells of the DZ, provided that if thetranscribed DNA region encodes a protein or polypeptide, or encodes anantisense RNA or ribozyme directed to a sense RNA encoded by anendogenous plant gene that is expressed in the plant in cells other thanthose of the DZ, the plant expressible promoter is a DZ-selectivepromoter, i.e., a promoter which directs expression of the transcribedregion selectively in cells of the DZ.

[0017] Preferably the transcribed DNA region encodes a protein orpolypeptide which is toxic to the cells in which it is produced, such asa barnase; a protein or polypeptide that increases the level of auxinsor auxin analogs in the cells in which it is produced, such as atryptophan monooxygenase and/or a indole-3-acetamide hydrolase; aprotein or polypeptide that increases the sensitivity to auxin in thecells in which it is produced, such as the roIB gene product; or aprotein or polypeptide that decreases the sensitivity to ethylene in thecells in which it is produced, such as the mutant ETR1-1 protein or aanother ethylene receptor protein.

[0018] In another preferred embodiment of this invention, thetranscribed DNA encodes an RNA, such as an antisense RNA or a ribozyme,part of which is complementary to the mRNA encoded by a gene which isnaturally expressed in the DZ, preferably a DZ-selective gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENTINVENTION

[0019] As used herein, the term “dehiscence” refers to the processwherein a plant organ or structure, such as an anther or fruit, opens atmaturity along a certain line or in a definite direction, resulting inthe shedding of the content of said organ or structure. In some of itsaspects the process of dehiscence is reminiscent of the process ofabscission, wherein a part or organ, such as a leaf, flower or fruit, isseparated from the rest of the plant.

[0020] As used herein, the term “pod” means a dry dehiscent fruit thatconsists of one, two or more carpels. In oilseed rape the pod is abivalve silique, wherein the valves are delineated by longitudinaldorsal and ventral sutures, which comprise the dehiscence zones.

[0021] As used herein, the term “pod dehiscence” means the processwherein a fruit, particularly a pod, splits open along a discrete layerof cells, eventually resulting in the separation of the valves andsubsequent shedding of the seeds contained within the fruit,particularly the pod. Pod dehiscence occurs in a large variety of plantsthat develop dry fruits, such as in most genera of the Cruciferae.

[0022] The term “dehiscence zone” (DZ) in its most general senseincludes the tissues in the zone along which a plant organ or structuresplits open during the process of dehiscence. Macroscopically the DZ canusually be recognized by the presence of a clear suture in the organ. Inthe strict sense the DZ may comprise a region of only 1-3 parenchymatouscells wide. In a pod, this region usually comprises densely packed cellsand is adjacent to the periphery of vascular tissue of the replumseparating it from the valve edges. For the purpose of this inventionthe DZ may also include the cell layers surrounding this region. The DZextends from the locule of the pod to the epidermal suture.

[0023] As used herein, the term “promoter” denotes any DNA which isrecognized and bound (directly or indirectly) by a DNA-dependentRNA-polymerase during initiation of transcription. A promoter includesthe transcription initiation site, and binding sites for transcriptioninitiation factors and RNA polymerase, and can comprise various othersites (e.g., enhancers), at which gene regulatory proteins may bind.

[0024] As used herein, the term “plant-expressible promoter” means apromoter which is capable of driving transcription in a plant cell. Thisincludes any promoter of plant origin, but also any promoter ofnon-plant origin which is capable of directing transcription in a plantcell, i.e., certain promoters of viral or bacterial origin such as theCaMV 35S or the T-DNA promoters.

[0025] The term “regulatory region”, as used herein, means any DNA, thatis involved in driving transcription and controlling (i.e., regulating)the timing and level of transcription of a given DNA sequence, such as aDNA coding for a protein or polypeptide. For example, a 5′ regulatoryregion (or promoter region) is a DNA sequence located upstream (i.e.,5′) of a coding sequence and which comprises the promoter and the5′-untranslated leader sequence. A 3′ regulatory region is a DNAsequence located downstream (i.e., 3′) of the coding sequence and whichcomprises suitable transcription termination (and/or regulation)signals, including one or more polyadenylation signals.

[0026] As used herein, the term “cell wall hydrolase” means an enzymethat is involved in the degradation of cell wall material, e.g., duringthe process of dehiscence. Examples of such enzymes include, but are notlimited to, polygalacturonase, cellulase (beta-1,4-glucanase),beta-galactosidase, proteases hydrolyzing cell wall proteins, and thelike.

[0027] The term “gene” means any DNA fragment comprising a DNA region(the “transcribed DNA region”) that is transcribed into a RNA molecule(e.g., a mRNA) in a cell under control of suitable regulatory regions,e.g., a plant expressible promoter. A gene may thus comprise severaloperably linked DNA fragments such as a promoter, a 5′ untranslatedleader sequence, a coding region, and a 3′ untranslated regioncomprising a polyadenylation site. An endogenous plant gene is a genewhich is naturally found in a plant species. A chimeric gene is any genewhich is not normally found in a plant species or, alternatively, anygene in which the promoter is not associated in nature with part or allof the transcribed DNA region or with at least one other regulatoryregions of the gene.

[0028] The term “expression of a gene” refers to the process wherein aDNA region under control of regulatory regions, particularly thepromoter, is transcribed into an RNA which is biologically active i.e.,which is either capable of interaction with another nucleic acid orwhich is capable of being translated into a biologically activepolypeptide or protein. A gene is said to encode an RNA when the endproduct of the expression of the gene is biologically active RNA, suchas an antisense RNA or a ribozyme. A gene is said to encode a proteinwhen the end product of the expression of the gene is a biologicallyactive protein or polypeptide.

[0029] The phenotypic effect of expression of a gene refers to thebiochemical, physiological and/or developmental effects of theproduction of the RNA or protein, encoded by the gene, on the plantcells (or plants) in which it is produced. Phenotypic effects of geneexpression may be reduced or prevented by reducing or preventing theproduction of the encoded RNA or protein, or by otherwise interferingwith the biological activity of such RNA or protein.

[0030] As defined herein, whenever it is stated in the specificationthat a “cDNA of such mRNA comprises the nucleotide sequence of SEQ IDNO: X” the RNA thus has the same nucleotide sequence as represented inSEQ ID NO: X except that the U-residues (in the RNA sequence) arereplaced by T-residues (in the DNA sequence).

[0031] In accordance with this invention, DZ-selective cDNAs and theircorresponding plant genomic DNA fragments are identified as follows:

[0032] 1) a cDNA library is constructed starting from mRNA isolated fromDZ tissue and the cDNA library is subjected to differential screening inorder to identify an mRNA which is selectively present in tissues of aparticular DZ when compared to other plant tissues including but notlimited to: pod walls, seeds, replum, leaves, stems, roots, reproductiveorgans, and the like. Alternatively, the cDNA library is screened witholigonucleotides, that are deduced from a determined amino acid sequenceof an isolated protein, such as, for example, a cell wall hydrolase,that is identified to be selectively present in the DZ. Furthermore, itis possible to use the same oligonucleotides in a nested-PCR approachand to use the amplified fragment(s) as a probe to screen the library.The DZ-selective cDNA library can be constructed from a pool of mRNAs,isolated at different stages of DZ development;

[0033] 2) a cDNA, encoding the DZ-selective mRNA or protein, is isolatedand characterized;

[0034] 3) this cDNA is used as a probe to identify and isolate theregion in the plant genome, comprising the nucleotide sequence encodingthe DZ-selective mRNA or protein. Alternatively, the genomic DNA can beisolated utilizing inverse PCR using oligonucleotides deduced from thecDNA sequence; and

[0035] 4) optionally, RNA probes corresponding to the cDNAs areconstructed and used in conventional RNA-RNA in-situ hybridizationanalysis [see e.g., De Block et al. (1993), Anal. Biochem. 215: 86] ofdifferent plant tissues, including the particular DZ of interest, toconfirm the selective presence of the mRNA produced by the presumedDZ-selective endogenous plant gene in that DZ.

[0036] The term “dehiscence zone-selective”, with respect to theexpression of a DNA in accordance with this invention, refers to, forpractical purposes, the highly specific, preferably exclusive,expression of a DNA in cells of one particular DZ, particularly a podDZ, or a limited series of DZs.

[0037] Thus a DZ-selective gene is an endogenous gene of a plant that isselectively expressed in the cells of certain dehiscence zones,particularly in the cells of the pod dehiscence zone of the plant. Anyplant which possesses the DZ of interest may be used for the isolationof DZ-selective genes. Suitable plants for the isolation of DZ-selectivegenes are plants of the family Cruciferae including but not limited toArabidopsis thaliana, Brassica campestris, Brassica juncea, andespecially Brassica napus; plants of the family Leguminosae includingbut not limited to Glycine max, Phaseolus vulgaris and the like. ThemRNA (or the cDNA obtained thereof) transcribed from such a gene is aDZ-selective mRNA (or cDNA). A promoter that drives and controls thetranscription of such a mRNA is referred to as a DZ-selective promoter.A DZ-selective promoter can for instance be used to express a cytotoxicgene (e.g., a barnase gene) in a plant so that normal growth anddevelopment, and agronomical performance (as measured for instance byseed yield) of the plant is not negatively affected by expression of thecytotoxic gene in cells other than the DZ cells, preferably in cellsother than the pod DZ cells.

[0038] Once the DZ-selective gene (i.e., the genomic DNA fragment,encoding the DZ-selective mRNA from which the DZ-selective cDNA can beprepared) is obtained, the promoter region containing the DZ-selectivepromoter is determined as the region upstream (i.e., located 5′ of fromthe codon coding for the first amino acid of the protein encoded by themRNA. It is preferred that such promoter region is at least about 400 to500 bp, preferably at least about 1000 bp, particularly at least about1500 to 2000 bp, upstream of the start codon. For convenience, it ispreferred that such promoter region does not extend more than about 3000to 5000 bp upstream of the start codon. The actual DZ-selective promoteris the region of the genomic DNA upstream (i.e., 5′) of the regionencoding the DZ-selective mRNA. A chimeric gene comprising aDZ-selective promoter operably linked to the coding region of the qusgene [Jefferson et al. (1986), Proc. Natl. Acad. Sci. USA 83: 8447] willselectively produce, in transgenic plants, detectable beta-glucuronidaseactivity (encoded by the qus gene)in the cells of the particular DZ ofinterest, as assayed by conventional in-situ histochemical techniques[De Block and Debrouwer (1992), The Plant Journal 2: 261; De Block andDebrouwer (1993), Planta 189: 218].

[0039] Preferred DZ-selective genes from which DZ-selective promoterscan be obtained, are genes, preferably Brassica napus genes, that encodea DZ-selective mRNA from which a cDNA can be prepared that contains thesequence corresponding to the sequence of oligonucleotide PG1 (SEQ IDNO: 3) between nucleotide positions 11 and 27 and/or the sequence ofoligonucleotide PG3 (SEQ ID NO: 5) between nucleotide positions 11 and27(i.e. starting at position 11 and ending at position 27); and/orcontains the sequence complimentary to the oligonucleotide PG2 (SEQ IDNO: 4) between nucleotide positions 11 and 25 and/or the sequence of theoligonucleotide PG5 (SEQ ID NO: 6) between nucleotide positions 11 and27. Preferably, such DZ-selective cDNA contains aforementioned sequencesof oligonucleotides PG1 and PG3 and PG2 and PG5, or encodes a proteinencoded by the region of SEQ ID NO: 1 between nucleotide positions 95and 1,393.

[0040] A particularly preferred DZ-selective gene is the Brassica napusgene that encodes a DZ-selective mRNA from which a cDNA can be preparedthat contains the sequence of SEQ ID NO: 1 at least between nucleotides10 and 1600. Another preferred DZ-selective gene is the Brassica napusgene, that encode a DZ-selective mRNA from which a cDNA can be preparedthat contains the sequence of SEQ ID NO: 11.

[0041] A preferred promoter of the present invention is the promotercontained in the 5′ regulatory region of a genomic clone correspondingto the cDNA of SEQ ID NO: 1, e.g. the 5′ regulatory region with thesequence of SEQ ID NO: 14 starting at position 1 and ending at position2,328. A convenient promoter region is the DNA fragment comprising thesequence of SEQ ID NO: 14 starting anywhere between the unique SphI site(positions 246-251) and the HindII site (positions 1,836-1,841),particularly between the SphI site and the BamHI site (positions1,051-1,056), and ending at nucleotide position 2,328 Oust before theATG translation start codon). Such a promoter region comprises theDZ-selective promoter of the subject invention and the 5′ untranslatedleader region and is used for the construction of DZ-selective chimericgenes. In this regard a particular useful promoter region is the DNAfragment (hereinafter referred to as “PDZ”) with the sequence of SEQ IDNO: 14 between positions 251 (the SphI site) and 2,328.

[0042] However, smaller DNA fragments can be used as promoter regions inthis invention and it is believed that any fragment of the DNA of SEQ IDNO: 14 which contains at least the about 490 basepairs, more preferablyat least about 661 basepairs and most preferably about 1326 basepairs,upstream from the translation inititation codon can be used.Particularly preferred smaller fragments to be used as promoter regionin this invention have a DNA sequence comprising the sequence of SEQ IDNO: 14 between nucleotides 1002 and 2328.

[0043] It is assumed that DZ-specificity of the promoter of the 5′regulatory region of SEQ ID NO: 14 can be considerably improved byinclusion of the nucleotide sequence of SEQ ID NO: 14 betweennucleotides 1002 and 1674. Therefore promoters comprising thisnucleotide sequence are particularly preferred.

[0044] Alternatively, artificial promoters can be constructed whichcontain those internal portions of the promoter of the 5′ regulatoryregion of SEQ ID NO: 14 that determine the DZ-selectivity of thispromoter. These artifical promoters can contain a “core promoter” or“TATA box region” of another promoter capable of expression in plants,such as a CaMV 35S “TATA box region” as described in WO 93/19188.Suitable promoter fragments or artificial promoters can be identified,for example, by their approriate fusion to a reporter gene (such as thegus gene) and the detection of the expression of the reporter gene inthe appropriate tissue(s) and at the appropriate developmental stage. Itis known that such smaller promoters and/or artificial promoterscomprising those internal portions of the 5′ regulatory region of SEQ IDNO:. 14 that determine the DZ selectivity can provide better selectivityof transcription in DZ-specific cells and/or enhanced levels oftranscription if the transcribed regions of the DZ-selective chimericgenes of the invention.

[0045] Besides the actual promoter, the 5′ regulatory region of theDZ-selective gene of this invention also comprises a DNA fragmentencoding a 5′ untranslated leader (5′UTL) sequence of an RNA locatedbetween the transcription start site and the translation start site. Itis believed that the 5′ transcription start site is located betweenposition 2,219 and 2,227 (in SEQ ID NO: 14), resulting in a 5′UTL ofabout 102 to 110 nucleotides in length. It is also believed that thisregion can be replaced by another 5′UTL, such as the 5′UTL of anotherplant-expressible gene, without substantially affecting the specificityof the promoter.

[0046] Other useful DZ-selective genes or cDNAs for use in thisinvention are those isolated from other sources, e.g., from othercultivars of B. napus or even from other plant species, for instance byusing the cDNA of SEQ ID NO: 1 (or SEQ ID NO: 11) as a probe to screengenomic libraries under high stringency hybridization conditions usingconventional methods as described in Nucleic Acid Hybridization: APractical Approach (1985), IRL Press Ltd UK (Eds. B. D. Hames and S. J.Higgins). A useful gene for the purpose of this invention is thus anygene characterized by encoding a mRNA from which a cDNA variant can beprepared that contains a coding region with a nucleotide sequence thatis essentially similar to that of the coding region of the cDNA clone ofSEQ ID NO: 1, and coding for a protein with polygalacturonase activity.Also promoter regions and promoters can be identified, for example,using such cDNA variants, which are essentially similar to a promoterregion or promoter with a sequence as contained in SEQ ID NO: 14.

[0047] With regard to nucleotide sequences (DNA or RNA), such assequences of cDNAs or of regulatory regions of a gene, “essentiallysimilar” means that when two sequences are aligned, the percent sequenceidentity—i.e., the number of positions with identical nucleotidesdivided by the number of nucleotides in the shorter of the twosequences—is higher than 80%, preferably higher than 90%, especiallywith regard to regulatory regions. The alignment of the two nucleotidesequences is performed by the Wilbur and Lipmann algorithm [Wilbur andLipmann (1983), Proc. Nat. Acad. Sci. U.S.A. 80: 726] using awindow-size of 20 nucleotides, a word length of 4 nucleotides, and a gappenalty of 4. Two essentially similar cDNA variants will typicallyencode proteins that are essentially similar to each other. For example,a variant of the cDNA of SEQ ID NO: 1 will typically encode a proteinwith an amino acid sequence which is essentially similar to the aminoacid sequence of the protein encoded by the cDNA of SEQ ID NO: 1. Withregard to “amino acid sequences”, essentially similar means that whenthe two relevant sequences are aligned, the percent sequenceidentity—i.e., the number of positions with identical amino acidresidues divided by the number of residues in the shorter of the twosequences—is higher than 80%, preferably higher than 90%. The alignmentof the two amino acid sequences is performed by the Wilbur and Lipmannalgorithm [Wilbur and Lipmann (1983), Proc. Nat. Acad. Sci. U.S.A. 80:726] using a window-size of 20 amino acids, a word length of 2 aminoacids, and a gap penalty of 4. Computer-assisted analysis andinterpretation of sequence data, including sequence alignment asdescribed above, can be conveniently performed using the programs of theIntelligenetics™ Suite (Intelligenetics Inc., CA).

[0048] In accordance with this invention, the DZ-selective cDNAs andgenomic DNAs, as well as the regulatory regions obtained from thegenomic DNAs are used to modify the dehiscence properties in plants,particularly pod dehiscence properties in Brassica napus.

[0049] Thus, in accordance with this invention, a recombinant DNA isprovided which comprises at least one DZ-selective chimeric genecomprising a plant expressible promoter and a transcribed DNA region,one or both of which is derived from a DZ-selective gene of thisinvention.

[0050] Expression of a DZ-selective chimeric gene in a transgenic plantwill have phenotypic effects only in the cells of the DZ. Thus,expression of a DZ-selective gene may selectively prevent, suppress,inhibit or reduce the phenotypic effects of expression of endogenousplant genes in a certain dehiscence zone (such as a pod DZ), mayselectively kill or disable cells of the dehiscence zone, or mayinterfere with the normal metabolism of DZ cells, resulting in the delayor prevention of dehiscence, particularly pod dehiscence. For thepurpose of this invention, a plant cell (such as a DZ cell) is killed ordisabled if either all biochemical and/or physiological processes of thecell are stopped or, alternatively, if the biochemical and/orphysiological processes of the cell are changed to effectively reducethe extracellular production of at least one enzyme involved in thedegradation of plant cell walls, particularly a pectin degrading enzymesuch as a polygalacturonase, preferably by at least 30%, particularly byat least 75%, more particularly by at least 90%.

[0051] For the purpose of the present invention, the phenotypic effectsof expression of an endogenous gene in a plant cell is prevented,suppressed, inhibited or reduced if the amount of mRNA and/or proteinproduced by the cell by expression of the endogenous gene is reduced,preferably by at least 30%, particularly by at least 75%, moreparticularly by at least 90%.

[0052] Plants, in which dehiscence is delayed to different extents, oreven prevented, are produced by transforming a plant with a recombinantDNA comprising at least one DZ-selective chimeric gene of this inventionwhose expression in the plant results in the production of RNA or aprotein or polypeptide which interferes to different degrees with thenormal functioning of the cells of the dehiscence zone, for example, byreducing the phenotypic effects of expression of one or more endogenousgenes that encode cell wall hydrolytic enzymes, or by killing the DZcells. A delay in the onset of dehiscence, particularly fruitdehiscence, whereby pre-harvest shattering of seeds can be reduced orprevented, will find its application in those plants that suffer frompre-mature (i.e., prior to harvest) seed loss.

[0053] In a preferred embodiment of the present invention theDZ-selective chimeric gene comprises a transcribed DNA region which istranscribed into an RNA the production of which in the cells of the DZreduces, inhibits or prevents the expression of an endogenous gene,preferably a gene encoding a cell wall hydrolase, particularly anendo-polygalacturonase, in the cells of the DZ. The reduction of theexpression of the endogenous gene can be demonstrated by the reductionof the cytoplasmic levels of the mRNA normally produced by theendogenous gene. The endogenous gene as isolated from the plant willhereinafter be designated as the sense gene which encodes a sense mRNA(or sense pre-mRNA, i.e., an unprocessed mRNA which may include intronregions).

[0054] It is preferred that the endogenous sense gene encodes an enzymeinvolved in cell wall hydrolysis, preferably a pectin-degrading enzyme,such as a pectin esterase, a pectin methyl esterase, a pectin lyase, apectate lyase, a polygalacturonase and the like, and particularly anendo-PG. It is believed that pectin degrading enzymes, particularlyendo-polygalacturonases, play an important role in the degradation ofthe middle lamella material of plant cell walls and in the process ofdehiscence, and that selective inhibition of the production of suchenzymes in the dehiscence zone or in the region surrounding thedehiscence zone (e.g., by expression of an antisense RNA to the endo-PGencoding mRNA) on the average delays pod shatter for at least 1 day,preferably 2-5 days.

[0055] Although the sense gene may encode any cell wall hydrolase, thatis secreted by the cells of the DZ during the process of dehiscence, andthat is involved in the degradation of cell wall material in a certaindehiscence zone, such as for example a cellulase, a glucanase, or abeta-galactosidase, it is preferred that the sense gene is an endogenousDZ-selective gene.

[0056] Thus, in one aspect of this invention the DZ-selective chimericgene of this invention encodes an antisense RNA which is complementaryto at least part of a sense mRNA or sense pre-mRNA. Such antisense RNAis said to be directed to the sense RNA (or sense pre-mRNA). In thisregard, the encoded antisense RNA comprises a region which iscomplementary to a part of the sense mRNA or sense pre-mRNA, preferablyto a continuous stretch thereof of at least 50 bases in length,preferably of at least between 100 and 1000 bases in length. The upperlimit for the length of the region of the antisense RNA which iscomplementary to the sense RNA is of course the length of thefull-length sense pre-mRNA, or to the full length sense mRNA (which mayor may be not processed from a sense pre-mRNA), produced by the plantcells can be used. However, the antisense RNA can be complementary toany part of the sequence of the sense pre-mRNA and/or of the processedsense mRNA: it may be complementary to the sequence proximal to the 5′end or capping site, to part or all of the 5′ untranslated region, to anintron or exon region (or to a region bridging an exon and intron) ofthe sense pre-mRNA, to the region bridging the noncoding and codingregion, to all or part of the coding region including the 3′ end of thecoding region, and/or to all or part of the 3′ untranslated region. Incase the sense gene is a member of a gene family, it is preferred thatthe antisense RNA encoded by the DZ-selective chimeric gene of thisinvention contains a sequence which is complementary to a region of thesense RNA, e.g., a DZ-selective sense RNA, of at least 50 nucleotidesand which has a percent sequence identity (see above) of less than 50%,preferably less than 30%, with any region of 50 nucleotides of any senseRNA encoded by any other member of the gene family.

[0057] The transcribed DNA region in the DZ-selective chimeric gene ofthis invention can also encode a specific RNA enzyme, or so-calledribozyme (see, e.g., WO89/05852), capable of highly specific cleavage ofthe sense mRNA or sense pre-mRNA. Such ribozyme is said to be directedto the sense RNA (or sense pre-mRNA).

[0058] Expression of the endogenous gene producing a sense mRNA in aplant can also be inhibited or repressed by a DZ-selective chimeric genewhich encodes part or all, preferably all, of such sense RNA [Jorgensenet al. (1992), AgBiotech News Info 4: 265N].

[0059] The sense RNA to which the antisense RNA or ribozyme encoded bythe DZ-selective chimeric gene of this invention is directed ispreferably a mRNA, wherein a (doublestranded) cDNA of such mRNAcomprises the nucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO: 11) orvariants thereof. A preferred region of the sense RNA to which theantisense RNA or ribozyme encoded by the DZ-selective chimeric gene ofthis invention is directed comprise a nucleotide sequence of SEQ ID NO:.1 starting anywhere between nucleotide 890 and 950 and ending anywherebetween nucleotide 1560 and 1620. Another preferred region of the senseRNA to which the antisense RNA or ribozyme encoded by the DZ-selectivechimeric gene of this invention is directed comprise a nucleotidesequence of SEQ ID NO:. 1 starting anywhere between nucleotide 1280 and1340 and ending anywhere between nucleotide 1560 and 1620, such as, butnot limited to, the nucleotide sequence between nucleotides 1296 and1607.

[0060] A DZ-selective chimeric gene encoding a antisense RNA orribozyme, as described above, is preferably under the control of aDZ-selective promoter. Particularly useful DZ-selective promoters arethe promoters from the DZ-selective genes described above, particularlythe promoter as conained within the 5′ regulatory region of SEQ ID NO:14 between position 1 and 2,328. However, if the DZ-selective geneencodes an antisense RNA and/or ribozyme which is directed to a senseRNA produced by an endogenous DZ-selective gene, preferably a geneencoding a endo-polygalacturonase, it is not required that the promoterof the DZ-selective chimeric gene be a DZ-selective promoter.Nevertheless, in such case the promoter of the DZ-selective gene shoulddirect expression at least in cells of the DZ. Indeed, because the senseRNA is produced selectively in the cells of the DZ, the production ofthe antisense RNA or ribozyme encoded by the DZ-selective gene in cellsother than the cells of the DZ, will not have a noticeable phenotypiceffect on such cells. Examples of promoters that direct expression atleast in cells of the DZ are constitutive plant expressible promoterssuch as the promoter (P35S) of the 35S transcript of Cauliflower mosaicvirus (CaMV)[Guilley et al. (1982), Cell 30: 763], or the promoter(Pnos) of the nopaline synthase gene of Agrobacterium tumefaciens[Depicker et al. (1982), J. Mol. AppL Genet. 1: 561].

[0061] In another preferred embodiment of this invention, theDZ-selective chimeric gene encodes an mRNA which, when produced in plantcells, is translated into a protein or polypeptide which interferes withthe metabolism and/or physiology of the plant cells. In most casesproduction of such protein or polypeptide will be undesired in cellsother than the DZ cells and in this regard it is preferred that suchchimeric genes comprise a DZ-selective promoter. Particular usefulDZ-selective promoters are again the promoters from the DZ-selectivegenes described above.

[0062] In one aspect of this invention the DZ-selective chimeric gene ofthis invention comprises a transcribed DNA region encoding a protein theactivity of which will result in an increase in biologically activeauxins or auxin analogs within the cells. Such protein may for instancebe involved in auxin biosynthesis, such as tryptophan monooxygenaseand/or the indole-3-acetamide hydrolase, encoded by the Agrobacteriumtumefaciens T-DNA gene 1 (iaaM) and/or gene 2 (iaaH), respectively[Gielen et al. (1984), The EMBO J. 3: 835], or may be theamidohydrolase, encoded by the Arabidopsis thaliana ILR1 gene, whichreleases active indole-3-acetic acid (IAA) from IAA-conjugates [Barteland Fink (1995), Science 268: 1745]. In view of the observed decline inIAA levels prior to pod dehiscence (see Example 1), it is believed thatproduction of such auxin increasing proteins selectively in the DZ cellsof a plant, will not result in the killing of the cells due tooverproduction of IAA, but will rather result in the maintenance and/orrestoration of the IAA levels substantially as found before the observeddecline. This delays the onset of pod dehiscence, through a prolongedinhibition by IAA of production and/or activity of cell wall hydrolyticenzyme normally produced by the cells in the dehiscence zone.

[0063] Alternatively the transcribed DNA region of the DZ-selectivechimeric gene of this invention can comprise the open reading frame ofthe Agrobacterium rhizogenes roIB gene [Furner et al. (1986), Nature319: 422]. Expression of such DZ-selective chimeric gene in a plant willresult in an increase of the sensitivity of the plant cells towardsauxin through the production of the roIB gene product in cells of thepod DZ thereby countering the normal decline in IAA concentration in theDZ prior to pod shattering.

[0064] In another aspect of the present invention, the DZ-selectivechimeric gene of this invention comprises a transcribed DNA regionencoding a protein, the activity of which results in a decrease of thesensitivity towards ethylene of the plant cells in which it is produced.Indeed, several genes involved in the ethylene signal transductionpathway in plants have been identified by mutational analysis (e.g.ETR1, ETR2, EIN4, ERS, CTR1, EIN2, EIN3, EIN5, EIN6, HLS1, EIR1, AUX1,EIN7) and for a number of them, the corresponding genes have been cloned[Chang (1996), TIBS 21:129; Bleecker and Schaller (1996), Plant Physiol111:653]. It is thought that ETR1, ETR2, EIN4, ERS all encode ethylenereceptors, while the rest of the genes would be involved in the ethylenesignal transduction pathway downstream of the receptors [Ecker (1995),Science 268: 667]. The ethylene receptors which have been sequenced,bear homology to the receiver domain of the response regulator componentand/or to the histidine protein kinase domain of the sensor component ofthe so-called bacterial two-component regulators and are divided in twoclasses according to the presence or absence of the receiver domainhomology. Class I ethylene receptors comprise both the sensor andreceiver homologous domains and are exemplified by ETR1 (Arabidopsis),and eTAE1 (tomato). Class II ethylene receptors comprise only the domainhomologous to the histidine protein kinase domain of the sensorcomponent and are exemplified by ERS (Arabidopsis) and NR (tomato).Receptors encoded by mutant alleles of the identified genes confer adominant insensitivity to ethylene [Chang (1996), supra; Bleecker andSchaller (1996), supra]. Therefore an example of a DZ-selective chimericgene, comprising a transcribed DNA region encoding a protein whoseactivity results in a decrease of the sensitivity towards ethylene ofthe plant cells in which it is produced, is one which comprises the openreading frame of a dominant, ethylene-insensitive, mutant allele of theArabidopsis thaliana ETR1 gene, such as ETR1-1 [Chang et al. (1993),Science 262: 539]. A plant in which such DZ-selective chimeric gene isexpressed produces a mutant ethylene receptor (the ETR1-1 protein)selectively in the cells of the DZ and these cells therefore becomeinsensitive towards the phytohormone ethylene and do not respond(metabolically) to changes in the concentration of the hormone, such asthe ethylene climacteric observed prior to the onset of pod dehiscence.It is thought that alternatively, a transcribed DNA region comprising anopen reading frame of a dominant, ethylene-insensitive, mutant allele ofany one of the mentioned class I ethylene receptors can be used to thesame effect. In another example of such a DZ-selective chimeric gene,conferring ethylene-insensitivity to the plants cells expressing theDZ-selective chimeric gene, a transcribed DNA region comprising an openreading frame of a dominant, ethylene-insensitive, mutant allele of anyone of the mentioned class II ethylene receptors, such as theArabidopsis thaliana ERS gene [Hua et al. (1995), Science 269: 1712] orthe tomato NR gene [Wilkinson et al. (1995), Science 270:1807] can beused for the same purpose.

[0065] It is further assumed that the rest of the products encoded bythe genes, involved in the ethylene signal transduction pathway,mentioned above, act downstream of the receptors. For CTR1, EIN2 andEIN3 the genes have been cloned [Ecker (1995), Science 268: 667].Modulation of the expression of the after genes in the dehiscence zone,e.g. by antisense RNA or ribozyme RNA, transcribed under control of aDZ-specific promoter, which is targetted towards the mentioned genes,will also influence sensitivity towards ethylene.

[0066] In another aspect of this invention the DZ-selective chimericgene of this invention comprises a transcribed DNA region encoding aprotein or polypeptide which, when produced in a plant cell, such as acell of a pod DZ, kills such cell or at least interferes substantiallywith its metabolism, functioning or development. Examples of suchtranscribed DNA regions are those comprising DNA sequences encodingribonucleases such as RNase T1 and especially barnase [Hartley (1988),J. Mol. Biol. 202: 913]; cytotoxins such as the A-domain of diphtheriatoxin [Greenland et al. (1983), Proc. Natl. Acad. Sci. USA 80: 6853] orthe Pseudomonas exotoxin A. Several other DNA sequences encodingproteins with cytotoxic properties can be used in accordance with theirknown biological properties. Examples include, but are not limited to,DNA sequences encoding proteases such as papain; glucanases; lipasessuch as phospholipase A2; lipid peroxidases; methylases such as the E.coli Dam methylase; DNases such as the EcoRI endonuclease; plant cellwall inhibitors, and the like.

[0067] In still another aspect of this invention the DZ-selectivechimeric gene of this invention comprises a transcribed DNA regionencoding a protein or polypeptide which is capable of being secretedfrom plant cells and of inhibiting at least the activity of at least oneendo-polygalacturonase that is produced in a dehiscence zone (such as apod DZ), particularly the endo-PG encoded by the cDNA of SEQ ID NO: 1.

[0068] In the DZ-selective chimeric gene of this invention it ispreferred that the 5′ untranslated region of encoded RNA is normallyassociated with the promoter, such as a DZ-selective promoter, of thechimeric gene. However, the 5′ untranslated region may also be fromanother plant expressible gene. Thus, it is preferred that aDZ-selective chimeric gene of this invention comprises the complete 5′regulatory region (including the 5′ untranslated region) of aDZ-selective gene. A particularly useful 5′ regulatory region is aregion of SEQ ID NO: 14, immediately upstream of position 1,329,preferably a region of at least 490 bp, more preferably a regionextending to the first SphI site upstream of position 2,329.

[0069] The DZ-selective chimeric genes of this invention preferably alsocomprise 3′ untranslated regions, which direct correct polyadenylationof mRNA and transcription termination in plant cells. These signals canbe obtained from plant genes such as polygalacturonase genes, or theycan be obtained from genes that are foreign to the plants. Examples offoreign 3′ transcription termination and polyadenylation signals arethose of the octopine synthase gene [De Greve et al. (1982), J. Mol.Appl. Genet 1: 499], of the nopaline synthase gene [Depicker et al.(1982), J. Mol. Appl. Genet. 1: 561] or of the T-DNA gene 7 [Velten andSchell (1985), Nucl. Acids Res. 13: 6998] and the like.

[0070] Preferably, the recombinant DNA comprising the DZ-selectivechimeric gene also comprises a conventional chimeric marker gene. Thechimeric marker gene can comprise a marker DNA that is; under thecontrol of, and operatively linked at its 5′ end to, a plant-expressiblepromoter, preferably a constitutive promoter, such as the CaMV 35Spromoter, or a light inducible promoter such as the promoter of the geneencoding the small subunit of Rubisco; and operatively linked at its 3′end to suitable plant transcription termination and polyadenylationsignals. The marker DNA preferably encodes an RNA, protein orpolypeptide which, when expressed in the cells of a plant, allows suchcells to be readily separated from those cells in which the marker DNAis not expressed. The choice of the marker DNA is not critical, and anysuitable marker DNA can be selected in a well known manner. For example,a marker DNA can encode a protein that provides a distinguishable colorto the transformed plant cell, such as the A1 gene (Meyer et al. (1987),Nature 330: 677), can provide herbicide resistance to the transformedplant cell, such as the bar gene, encoding resistance tophosphinothricin (EP 0,242,246), or can provided antibiotic resistanceto the transformed cells, such as the aac(6′) gene, encoding resistanceto gentamycin (WO94/01560).

[0071] The DZ-selective promoters of this invention are believed to behighly specific in activity or effect with regard to directing geneexpression in cells of the DZ. However the characteristics (e.g.,tissue-specificity) of a promoter contained in a chimeric gene may beslightly modified in some plants that are transformed with such chimericgene. This can, for example, be attributed to “position effects” as aresult of random integration in the plant genome.

[0072] Therefore in some plants transformed with the DZ-selectivechimeric gene of this invention low-level expression of the chimericgene may be observed in certain non-DZ cells. Thus, optionally, theplant genome can also be transformed with a second chimeric genecomprising a second transcribed DNA region, that is under control of asecond plant-expressible promoter and that encodes a RNA, protein orpolypeptide which is capable of counteracting, preventing or inhibitingthe activity of the gene product of the DZ-selective chimeric gene. Ifthe DZ-selective chimeric gene encodes barnase it is preferred that thesecond chimeric gene encodes a barstar, i.e., an inhibitor of barnase[Hartley (1988), J. Mol. Biol. 202: 913]. Other useful proteins encodedby the second chimeric genes are antibodies or antibody fragments,preferably single chain antibodies, that are capable of specific bindingto the protein encoded by the DZ-selective chimeric gene whereby suchprotein is biologically inactivated.

[0073] Preferably the second promoter is capable of driving expressionof the second transcribed DNA region at least in non-DZ cells of theplant to counteract, prevent or inhibit the undesired effects of lowexpression of the DZ-selective chimeric gene in such cells in sometransformed plants. Examples of useful second promoters are the CaMVminimal 35S promoter [Benfey and Chua (1990), Science 250: 959] or thepromoter of the nopaline synthase gene of Agrobacterium tumefaciensT-DNA [Depicker et al. (1982), J. Mol. Appl. Genet. 1: 561]. Otheruseful promoters are promoters from genes that are known not to beactive in the DZ, such as Brassica napus genes encoding a mRNA fromwhich a cDNA can be prepared that comprises the sequence of SEQ ID. No8, SEQ ID NO: 10, or SEQ ID NO: 12.

[0074] In plants the second chimeric gene is preferably in the samegenetic locus as the DZ-selective chimeric gene so as to ensure theirjoint segregation. This can be obtained by combining both chimeric geneson a single transforming DNA, such as a vector or as part of the sameT-DNA. However, in some cases a joint segregation is not alwaysdesirable. Therefore both constructs can be present on separatetransforming DNAs, so that transformation might result in theintegration of the two constructs at different location in the plantgenome.

[0075] In still a further embodiment of the present invention, a plantwith modified dehiscence properties can be obtained from a single plantcell by transforming the cell in a known manner, resulting in the stableincorporation of a DZ-selective chimeric gene of the invention into thenuclear genome.

[0076] A recombinant DNA comprising a DZ-selective chimeric gene can bestably incorporated in the nuclear genome of a cell of a plant,particularly a plant that is susceptible to Agrobacterium-mediatedtransformation. Gene transfer can be carried out with a vector that is adisarmed Ti-plasmid, comprising a DZ-selective chimeric gene of theinvention, and carried by Agrobacterium. This transformation can becarried out using the procedures described, for example, in EP0,116,718. Ti-plasmid vector systems comprise a DZ-selective chimericgene between the T-DNA border sequences, or at least to the left of theright T-DNA border. Alternatively, any other type of vector can be usedto transform the plant cell, applying methods such as direct genetransfer (as described, for example, in EP 0,233,247), pollen-mediatedtransformation (as described, for example, in EP 0,270,356, WO85/01856and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation(as described, for example, in EP 0,067,553 and U.S. Pat. No.4,407,956), liposome-mediated transformation (as described, for example,in U.S. Pat. No. 4,536,475), and the like.

[0077] Other methods, such as microprojectile bombardment as described,for example, by Fromm et al. [(1990), Bio/Technology 8: 833] andGordon-Kamm et al. [(1990), The Plant Cell 2: 603], are suitable aswell. Cells of monocotyledonous plants, such as the major cereals, canalso be transformed using wounded or enzyme-degraded intact tissuecapable of forming compact embryogenic callus, or the embryogenic callusobtained thereof, as described in WO92/09696. The resulting transformedplant cell can then be used to regenerate a transformed plant in aconventional manner.

[0078] The obtained transformed plant can be used in a conventionalbreeding scheme to produce more transformed plants with the samecharacteristics or to introduce the DZ-selective chimeric gene of theinvention in other varieties of the same or related plant species. Seedsobtained from the transformed plants contain the DZ-selective chimericgene of the invention as a stable genomic insert.

[0079] The following Examples describe the isolation andcharacterization of a DZ-selective gene from Brassica napus, theidentification of DZ-selective promoter, and the use of such a promoterfor the modification of dehiscence properties in plants. Unless statedotherwise in the Examples, all recombinant DNA techniques are carriedout according to standard protocols as described in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel etal. (1994) Current Protocols in Molecular Biology, Current Protocols,USA. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK.

[0080] In the examples and in the description of the invention,reference is made to following sequences of the Sequence Listing: SEQ IDNO: 1: DZ-selective cDNA encoding a endo-polygalacturonase of Brassicanapus SEQ ID NO: 3: oligonucleotide PG1 SEQ ID NO: 4: oligonucleotidePG2 SEQ ID NO: 5: oligonucleotide PG3 SEQ ID NO: 6: oligonucleotide PG5SEQ ID NO: 7: PCR Fragment BPG32-26 SEQ ID NO: 8: PCR Fragment KPG32-8SEQ ID NO: 9: PCR Fragment LPG12-16 SEQ ID NO: 10: PCR Fragment LPG32-24SEQ ID NO: 11: PCR Fragment LPG32-25 SEQ ID NO: 12: PCR FragmentLPG32-32 SEQ ID NO: 13: T-DNA of pGSV5 SEQ ID NO: 14: sequence ofgenomic clone comprising the DZ-selec- tive promoter region drivingexpression of an endopolygalacturonase gene of Brassica napus

[0081] In order to further illustrate the present invention andadvantages thereof, the following specific examples are given, it beingunderstood that the same are intended as illustrative and in nowiselimitative.

EXAMPLE 1

[0082] Characterization of Pod Dehiscence During Pod Development.

[0083] Endogenous Phytohormone Profiles During Pod Development.

[0084]Brassica napus cv Fido plants were grown in an unheatedgreenhouse. At 12 days after germination plants were transferred to, andfurther grown in, 1000 cm³ compost. Pods were collected at one weekintervals from two to eight weeks after anthesis. The pods were takenfrom the base of the terminal one of the first three axillary racemes.The pods were separated into dehiscence zone, pod wall and seeds.

[0085] The samples were grinded with a mortar and pestle and thenextracted for 16 hours at −20 C. in a total volume of 80% methanol.Purification and analysis of phytohormones was carried out essentiallyas described [Bialek and Cohen (1989), Plant Physiol. 90: 398; Prinsenet al. (1991), in: A Laboratory Guide for Cellular and Molecular PlantBiology. Ed. Negrutiu and Gharti-Chhetri. Birkhäuser Verlag,Basel/Boston/Berlin pp.175-185, pp.323-324; Chauvaux et al. (1993), J.Chromatogr. A 657: 337].

[0086] Different parts of the pods (pod wall, dehiscence zone and seeds)were screened for endogenous concentrations of the ethylene precursor1-aminocyclopropane-1-carboxylic acid (ACC) and conjugates thereof aswell as for indole-3-acetic acid (IAA) and conjugates thereof.

[0087] A peak in ethylene evolution [see also Meakin and Roberts (1990),J. Exp. Bot. 41: 1003] was observed immediately before pod shattering;this peak was correlated with observed peaks of free ACC. Especially inthe dehiscence zone a decline in IAA concentrations (free as well asconjugated forms) was observed, just before the onset of pod opening.This decline in IAA concentration was specifically correlated with anincreased cellulase activity in the dehiscence zone.

[0088] In a further experiment ethylene production was inhibited bytreating the pods with aminoethoxyvinylglycine (AVG), a competitiveinhibitor of the enzyme ACC-synthase. AVG was applied 28 days afteranthesis at 500 mg/l. This treatment resulted in a 40-50% reduction ofethylene production in the entire pod and was accompanied by a delay ofpod wall senescence of approximately 4 days. Decreased endogenous ACCconcentrations in both dehiscence zone and seeds of the treated podscorrelated with the reduced ethylene production. In the other tissuesanalysed (pod wall, septum and the zone between dehiscence zone and podwall) no such decrease in ACC concentrations or synthesis could bedemonstrated. A decrease in endogenous IAA content in the dehiscencezone preceding pod opening was also observed in these experiments bothin control and in AVG-treated plants.

[0089] To examine the auxin involvement in pod shattering, the syntheticauxin 4-chlorophenoxyacetic acid (4CPA) was used to manipulate auxinlevels. 4CPA was applied 35 days after anthesis as a spray at 150 mg/lin order to artificially keep auxin concentration at a high level duringthe entire period. This resulted in a distinct retardation in podshatter tendency (see Table 1), as well as a delay of pod wallsenescence of about two weeks. No effect was observed on the endogenousphytohormone concentrations. Beta-Glucanase activity however wasmarkedly decreased in the dehiscence zone. These results are clearlyindicative of the inhibitory effect of auxins on the production and/oractivity of beta-glucanase.

[0090] The decline in auxin is a major trigger of pod shatter. Table 1:Force (in 10⁻³N) needed to initiate and propagate pod opening asmeasured in the Cantilever bending test [Kadkol et al. [(1986), Aust. J.Bot. 34: 595] with pods (8% moisture) of oilseed rape cv Fido. Testedplants were either untreated, sprayed with AVG to reduce ethylenevalues, or sprayed with 4CPA to prevent the auxin drop. (SED: StandardError on Differences; df: degree of freedom) SED untreated AVG 4CPA (27df) To initiate 143.6 170.8 194.9 14.4  crack To propagate 167.1 171.4222.6 17.21 crack

[0091] Demonstration of Polygalacturonate-Degrading Enzyme Activity inPod Dehiscence Zones.

[0092] Pods of oilseed rape cv Fido were harvested at 6.5 weeks afteranthesis, stripped of the carpels and seeds, and crude enzyme extractswere prepared from tissues surrounding the dehiscence zones, includingthe replum with a vascular bundle and the thin membrane separating thetwo locules of the silique. Extracts were subsequently tested withrespect to their action against polymeric substrates (uronic acids),using molecular weight down-shift assays, based on gel-permeationchromatography of substrate incubated with a boiled (used as reference)and active enzyme preparation respectively. The assay in particulardetects enzymes with endo-activity as removal of single monosaccharidesin an exo-fashion only changes molecular weight distribution of thepolymeric substrate very slowly. Analysis for uronic acids was carriedout essentially as described by Blumenkrantz and Asboe-Hansen [(1973),Anal. Biochem. 54: 484]. The assay was used here only to demonstrate thepresence of enzyme activities in a strictly qualitative sense.

[0093] DZ preparations from oilseed rape pods contain all enzymeactivities required for a full depolymerization of pectic polymers oflow degree of methylation. It was found that one component of the enzymemixture was specifically acting on polygalacturonate polymers. It wasfurther demonstrated that only endo-polygalacturonase among known plantenzymes is responsible for the molecular weight down-shift of thepolygalacturonate preparations used.

[0094] It can be concluded that endo-polygalacturonase plays animportant role in the extensive degradation of middle lamella materialobserved during pod dehiscence.

[0095] Anatomical Observations During the Process of Dehiscence

[0096] Detailed examination of the structure of pod tissues has givenmore insight in the anatomical changes associated with the biochemicalprocesses that take place in the dehiscence zone. It was observed byelectron microscopy that rapid dehydration of the pod wall immediatelyprecedes the degradatio n of parenchymatous cells situated in thedehiscence zone, mesocarp, septum and in the seed abcission zone.Initial signs of breakdown were shown by swelling of the cell walls.Subsequent cell-separation was seen only in the dehiscence zone, and wasobserved to take place along the line of the middle lamella to befollowed by the dispersion of the microfibrils of the cell wall.Finally, all the cells of the dehiscence zone were observed to separatewhile the two valves of the pod remained attached only by the vascularstrands which pass through the dehiscence zone. Analysis using electronmicroscopy revealed very dramatic degradation of the middle lamelladuring pod opening while the primary cell wall was left essentiallyintact but for some thinning and softening processes. These observationsindicate that any processes in the primary cell wall are accessory tothe degradation of the middle lamella.

[0097] The complete dissolution of the middle lamella of cells in thedehiscence zone indicates the presence of pectin degrading enzymes suchas endoPG. While such enzymes degrade charged portions of the middlelamella pectins, other polysaccharide hydrolases, with affinity towardsneutral polymers, are involved to complete the depolymerization of themiddle lamella.

[0098] A beta-galactanase and a beta-glucanase were purified tohomogeneity. Detailed investigation of the substrate specificityindicated that these enzymes are involved in thinning of the primarycell wall in the deiscence zone.

EXAMPLE 2

[0099] Isolation of a DZ-Selective Endo-Polygalacturonase cDNA Clonefrom Brassica Napus.

[0100] Poly-A⁺ mRNA of pod dehiscence zones of Brassica napus cv Topazplants was prepared as follows. Twenty grams of tissue (leaves,dehiscence zones, pod walls, roots or stems) were ground in liquidnitrogen and homogenized for 30 seconds in a Waring blender with 100 mlof extraction buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate,pH 7.0, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol). The homogenate wastransferred to a fresh tube and 1/10 volume of 2 M sodium acetate, pH4.0, and 1 volume of TE saturated phenol/chloroform was added. Thesolution was shaked vigorously, cooled on ice for 15 min. andcentrifuged at 10,000×g for 15 min at 4 C. The supernatant wasre-extracted with phenol/chloroform as described above. An equal volumeof isopropanol was added to the re-extracted supernatant and RNA wasprecipitated by an overnight incubation at −20 C. After centrifugationat 10,000×g for 15 min, the RNA pellet was dissolved in 2 ml ofdenaturation buffer. Fourteen ml of 4 M LiCl was then added and thesolution kept in an ice-bath overnight. The RNA was pelleted bycentrifugation at 10,000×g for 15 min, washed in 80% ethanol, dried anddissolved in 1 ml of water. Poly-A⁺ RNA was isolated on an oligo-d(T)sepharose column according to the manufacturer's guidelines (Boehringer,Mannheim).

[0101] Random or oligo-d(T) primed first strand cDNA synthesis wasperformed using M-MLV reverse transcriptase and 6 μg of total poly-A⁺RNA as prepared above according to conditions outlined by themanufacturer (Life Technologies/BRL). First strand cDNAs were used astemplate DNA for further PCR reactions.

[0102] Four degenerated primers were designed based on conserved regionsfrom published polygalacturonase (PG) amino acid sequences from tomato[DellaPenna et al. (1986), Proc. Natl. Acad. Sci. USA 83:6420; Griersonet al. (1986), Nucl. Acids Res. 14: 8595], maize [Niogret et al (1991),Plant Mol. Biol. 17: 1155], avocado and Oenothera [Brown and Crouch(1990), The Plant Cell 2: 263]. The sequences of the four primers used(PG1, PG2, PG3 and PG5) are shown in SEQ ID NOS: 3-6. A restrictionenzyme site for EcoRI was introduced at the 5-end of the two upstreamprimers PG1 and PG3 and a BamHI site was introduced at the 5-end of thetwo downstream primers PG2 and PG5.

[0103] All PCR reactions had the following final composition: 50 mM KCl,10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl, and 0.001% (w/v) gelatin, 100pmoles of degenerated primers and 1U of Taq DNA polymerase in a 50 μlreaction volume. After an initial denaturation of template DNA at 95° C.for 3 minutes in 1×PCR reaction buffer, the PCR reaction was initiatedby adding 1U of Taq DNA polymerase in 1×PCR buffer (hot start PCR) usingthe following conditions: 1 min. at 95° C., 1 min. at 45° C. and 1 min.at 72° C. for 35 cycles followed by 72° C. for 3 min. For hot startnested PCR 2 μl of a PCR reaction was applied as template in a new PCRreaction. The PCR products were chloroform extracted and ethanolprecipitated, redissolved in TE and digested with the restrictionenzymes BamHI and EcoRI. The restricted PCR products were purified fromlow melting agarose, and cloned into pGEM-7z cut with BamHI and EcoRI.DNA sequences of the PCR fragments were obtained by the dideoxy chaintermination method using Sequenase version 2.0 (Pharmacia).

[0104] The longest PCR fragment was obtained by using the PG1/PG5 primercombination. Hot start nested PCR was performed with the PG3/PG2,PG1/PG2 or PG3/PG5 primer combinations using a small aliquot of thePG1/PG5 PCR reaction as a template. Seven highly divergent PG-relatedclones were identified by sequencing of the PCR products, indicating thepresence of at least seven different PG isoforms. Three forms wereobtained from a single tissue only, namely Ipg32-25 (SEQ ID NO: 11) fromdehiscence zones, kpg32-8 (SEQ ID NO: 8) from pod walls and bpg32-26(SEQ ID NO: 7) from leaves. Lpg32-32 (SEQ ID NO: 12), Ipg32-24 (SEQ IDNO: 10) were found only in the two pod tissues, whereas Ipg12-16 (SEQ IDNO: 9) was obtained from all three tissues analyzed. It should be noted,that Ipg35-8 (containing the DNA sequence of SEQ ID NO: 1 from position884 to 1,245) was the only type identified in the dehiscence zone whenthe PG3/PG5 primer combination was used in a nested PCR reaction.

[0105] The expression of the PG-related PCR clone Ipg35-8 in roots,stems, leaves and hypocotyls as well as during pod development wasinvestigated by Northern analysis as follows. Total RNA of individualtissues was separated by gel electrophoresis in 0.66 Mformaldehyde/1%agarose gel [Sambrook et al. (1989), supra]. RNA wastransferred onto Hybond-N filters and fixed to the filter byUV-irradiation. The filters were prehybridized for4 hours in 5×Denhardt,25 mM Na₂HPO₄, 25 mM NaH₂PO₄, 0.1% pyrophosphate, 750 mM NaCl, 5 mM EDTAand 100 μg/ml denatured herring sperm DNA at 68° C. The PCR productswere radioactively labelled and were heat-denatured and added directlyto the pre-hybridization buffer and hybridization was then continued for16 hours at 68° C. The filter was washed according to Sambrook et al.[(1989), supra] where the final wash was carried out at 68° C. in0.2×SSC, 0.1% SDS. The filters were autoradiographed at −80° C. using anintensifying screen.

[0106] No transcripts hybridizing to the Ipg35-8 clone could be detectedin total RNA isolated from roots, stems, leaves and hypocotyls However,the Ipg35-8 clone hybridized to a 1.6-1.7 kb transcript that isexclusively expressed in the dehiscence zone during all stages analyzedand was found to increase dramatically in abundance after week 5.

[0107] A DZ-selective cDNA library was constructed in Lambda ZAP® IIinsertion vectors (Stratagene) using 5 μg of poly-A⁺ RNA isolated fromdehiscence zones 6 weeks after anthesis. cDNAs larger than 1 kbp werepurified from a low temperature melting agarose gel and ligated into theLambda ZAP® II vector. The primary library consisted of 1.25×10⁶ pfuwith an averaged cDNA insert size of app. 1.5 kbp. Library screening wasdone according to standard procedures at high stringency [Sambrook et al(1989), supra].

[0108] cDNAs were sequenced using Sequenase v. 2.0 (Amersham). Sequenceanalysis was performed with the GCG sequence analysis software packagev. 7 [Devereux et al. (1984), Nucl. Acids Res. 12: 387].

[0109] Screening 300,000 plaques with the Ipg35-8 PCR-fragment as probegave approximately 200 positive hybridization signals. Five stronglyhybridizing plaques were purified to homogeneity. After excision of theinsert DNA from the lambda vector, restriction enzyme analysis showedthe cDNA inserts to be approximately 1600 bp in all cDNA clones exceptone, which only had an insert of 1300 bp. Restriction enzyme mapping ofthe 4 largest cDNA inserts (designated as X, 5, 9 and 11 respectively)showed minor differences between the 4 cDNA inserts.

[0110] Most noteworthy is the presence of a Nsil restriction enzyme sitein cDNA clones X and 11 and the presence of a HindII site in cDNA clone9. In contrast, none of these restriction sites are present in cDNAclone 5. Partial sequencing of the 5 and 3 cDNA ends revealed additionalsequence variations including small deletions/insertions between thedifferent cDNA clones. These results indicate the expression in thedehiscence zone of different but highly homologous PG-encoding genes.The sequence data also showed that the larger 4 cDNA inserts allcontained the complete coding sequence for the PG protein.

[0111] The complete sequence of cDNA clone X and the deduced amino acidsequence of its largest open reading frame is shown in SEQ ID NO: 1. Theopen reading frame encodes a protein of 433 amino acids in size with anestimated molecular weight of 46.6 kD and with considerable similarityto known endo-polygalacturonases. Similar to other cell wall hydrolasesthe presumed DZ-selective endo-PG is initially produced as a precursorcontaining a N-terminal signal peptide which is cleaved offco-translationally. The most likely cleavage site is located betweenamino acids 23 and 24 and gives rise to a mature protein with anestimated molecular weight of 44.2 kD.

[0112] Northern analysis, using cDNA clone X as a probe, confirmed andextended the previously obtained expression pattern. Total RNA wasprepared as described from different tissues of the pods (the dehiscencezone, the pod walls, seeds and septum) at 5 time points (2, 3, 5, 7 and9 weeks after anthesis—WAA). 5 μg of total RNA was seperated bygel-electrophoresis and hybridized with the radiolabelled cDNA of SEQ IDNO: 1 as a probe under the stringent conditions descibed above. Theautoradiogram was developed after overnight exposure. At 2 WAA, nosignal was detectable; at 3 WAA a faint signal was observed; based ondensitometry scannings and readings, the expression level measured attime point 5 WAA is about 3.5× the amount seen at 3 WAA, at 7 WAA isabout 7× the amount seen at 3 WAA, at 9 WAA is about 12 times the amountseen at 3 WAA. No signal was detected in the pod walls or seeds. Faintexpression (comparable with the level in the DZ at 3 WAA) was measuredin the septum at 9 WAA.

[0113] The RNA used in this experiment has been extracted from therespective tissues of plants for which the pod development took about 9weeks.

EXAMPLE 3

[0114] Isolation of a DZ-Selective Promoter from a B. napus GenomicClone Corresponding to the cDNA Clone Ipq 35-8.

[0115] A commercially available lambda EMBL3 Brassica napus cv. Bridgergenomic library (Clontech Laboratories, Inc.) in Escherichia coli strainNM538 was screened as follows. After transfer to Hybond-N nylonmembranes, the Ipg35-8 cDNA was radioactively labelled using randompriming, and the filters were hybridized under high stringencyconditions in 5×SSPE, 5×Denhardt, 0,5% SDS, 50 μg/ml herring DNA(1×SSPE: 0,18 M NaCl, 10 mM sodium phosphate, pH 7,7, 1 mM EDTA) andwashed under high stringency conditions (68° C., 0,1×SSPE, 0,1% SDS inthe final wash). Approximately 600,000 plaques were screened and elevenhybridizing plaques were isolated. Two hybridizing plaques, lambda 2 and11, were rescreened twice. Following the second rescreening phagelysates were made from lambda 2 and 11 on E. coli NM538 grown withoutmaltose. DNA preparations from lambda 2 and lambda 11 were digested withSalI, subjected to gel electrophoresis and transferred to Hybond-N nylonmembrane. Hybridization with the labelled Ipg35-8 CDNA clone, resultedin identical hybridization patterns for both clones. A stronglyhybridizing 6.3 kb SalI fragment was isolated from lambda 11 andinserted into pUC18, resulting in the master clone 6.3SaI. In order toconfirm 6.3SaI as corresponding to Ipg35-8, a sequencing primer wasdesigned enabling the determination of a DNA stretch encoding two uniqueamino acids present in Ipg35-8. Dideoxy sequencing by the Sanger methodconfirmed that the isolated genomic clone 6.3SaI was in this respectidentical to the Ipg35-8 cDNA. Restriction mapping of this clonedemonstrated that it covered the entire Ipg35-8 open reading frame andcontained moreover app. 100 to 200 bp of downstream sequence and app.3.5 kb of upstream sequence. The DNA sequence of a stretch of about 2.3kb (including the promoter, the 5′ untranslated region and the first 24nucleotides of the open reading frame) was determined and is presentedin SEQ ID NO: 14.

[0116] In view of the fact that the cDNA clone (SEQ ID NO: 1) and thegenomic clone (SEQ ID NO: 14) were isolated from different B. napuscultivars (resp. cv.Topaz and cv.Bridger), it was surprisingly foundthat upon alignment of both sequences the overlapping fragment displayed100% sequence identity.

[0117] The transcription start site of the DZ selective genecorresponding to the gene contained in the 6.3SaI clone is determinedusing generally known techniques such as primer extension analysis[Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, NY] or RACE-PCR [Innis etal. (1990) PCR Protocols: A Guide to Methods and Applications, AcademicPress Inc.]. The 5′UTL is believed to be located between positions 2,219and 2,227 of SEQ ID NO: 14.

[0118] Using well-established site-directed mutagenesis techniques[Ausubel et al. (1994), supra], the DNA sequence is modified to create aunique restriction enzyme (e.g., NcoI) recognition site around the ATGtranslation initiation codon of the coding sequence. This allows astraightforward fusion of the promoter region of the DZ-selective geneto a DNA sequence of interest to construct a DZ-selective chimeric geneof the invention. Using a unique restriction enzyme recognition sitelocated between 500 to 2000 base pairs upstream (i.e., 5′) of the uniquerestriction site surrounding the ATG translation initiation codon, awell defined DNA fragment is isolated, that is subsequently used as apromoter cassette, hereinafter referred to as PDZ, that directsDZ-selective expression in plants.

[0119] For example, a SphI-NcoI fragment (of about 2.08 kb), which iscapable of directing DZ-selective expression in plants, is thensubsequently used as a promoter cassette, hereinafter referred to asPDZ1.

[0120] A DZ-selective chimeric gene (PDZ or PDZ1-gus-3′nos) isconstructed comprising the following operably linked DNA fragments:

[0121] PDZ or PDZ1: the 5′ regulatory region comprising a DZ-selectivepromoter,

[0122] gus: a DNA fragment coding for beta-glucuronidase [Jefferson etal. (1986) Proc. Natl. Acad. Sci. USA 83: 8447];

[0123] 3′nos: the 3′ untranslated end comprising the polyadenylationsite of the nopaline synthase gene (“3′nos”)[Depicker et al. (1982), J.Mol. Appl. Genet. 1: 561].

[0124] A second promoter cassette which is capable of directingDZ-selective expression in plants, was obtained using well-establishedsite-directed mutagenesis techniques to modify the DNA sequence tocreate a unique restriction site immediately upstream of the ATGtranslation initiation codon of the coding sequence. For this purpose aSmaI site has been engineered, by changing the A-nucleotides of SEQ IDNO:. 14 at positions 2327 and 2328 into G-nucleotides . The SphI-SmaIfragment of about 2.1 kb, hereinafter referred to as promoter cassettePDZ2, was fused at the SmaI site upstream of the GUS coding region inthe plasmid pBI101 (Clontech Laboratories, Inc CA, USA), resulting in aplasmid (2.1guspgem7) carrying the chimeric PDZ2-gus-3′nos geneconstruct.

[0125] A chimeric selectable marker gene PSSU-bar-3′ocs was constructed[De Almeida et al. (1989), Mol. Gen. Genet. 218: 78]. It comprises thefollowing operably linked DNA fragments:

[0126] PSSU: the promoter region of Arabidopsis thalianaribulose-1,5-biphosphate carboxylase small subunit 1A encoding gene[Krebbers et al. (1988), Plant Mol. Biol. 11: 745),

[0127] bar: the region of the bar gene encoding phosphinothricin acetyltransferase[Thompson et al. (1987), The EMBO J. 6: 2519],

[0128] 3′ocs: a 3′ untranslated end comprising the polyadenylation siteof the octopine synthase gene [De Greve et al. (1983), J. Mol. Appl.Genet. 1: 499].

[0129] Alternatively, a PSSU-bar-3′g7 was constructed comprisingidentical fragments as the preceding chimeric selectable marker gene,except that the 3′ocs was replaced by the 3′ untranslated end comprisingthe polyadenylation site of the T-DNA gene 7 (3′g7; Velten and Schell(1985), Nucl. Acids Research, 13, 6981).

[0130] Both the DZ-selective chimeric gene (PDZ2-gus-3′nos; cloned as aHindIII-XhoI fragment of about 4.2 kb) and the chimeric marker gene(PSSU-bar-3′g7) were introduced into the polylinker located between theborder sequences of the T-DNA vector pGSV5, resulting in plasmid vectorpTCO155 carrying the PDZ2-qus-3′nos and pSsuAra-bar-3′g7 chimeric geneconstructs between the T-DNA border repeats. pGSV5 was derived fromplasmid pGSC1700 [Cornelissen and Vandewiele (1989), Nucl. Acids Res.17: 833] but differs from the latter in that it does not contain abeta-lactamase gene and that its T-DNA is characterized by the sequenceof SEQ ID NO: 13.

EXAMPLE 4

[0131] Construction of a Chimeric-Gene Carrying the Barnase CodingRegion Under Control of the Endo-PG Promoter.

[0132] A DZ-selective chimeric gene (PDZ-barnase-3′nos) is constructedcomprising the following operably linked DNA fragments:

[0133] PDZ: the 5′ regulatory region of Example 3, comprising aDZ-selective promoter,

[0134] barnase: a DNA fragment coding for barnase of Bacillusamyloliquefaciens [Hartley (1988), J. Mol. Biol. 202: 913],

[0135] 3′nos

[0136] Both the DZ-selective chimeric gene and the PSSU-bar-3′ocschimeric marker gene are introduced into the polylinker located betweenthe border sequences of the T-DNA vector pGSV5 of Example 4.

[0137] PDZ2-barnase-3′nos between T-DNA border repeats was constructedby replacing the TA29 promoter upstream of the barnase coding region inpTC099, by the PDZ2 promoter cassette. To this end the 2.1 kb (blunted)SphI-SmaI fragment comprising PDZ2 was fused with its SmaI site to theblunted NcoI site overlapping with the ATG-codon which had beenengineered at the 5′end of the coding sequence for the mature barnase inpTCO99, resulting in the plasmid vector pTPR1 carrying thePDZ2-barnase-3′nos chimeric gene between the T-DNA border repeats. TheT-DNA vector part of pTCO99 is derived from that of pGSV5 by insertionof an EcoRI linker (GGAATTCC) into the SmaI site of the polylinker, anda BgIII linker (CAGATCTG) into the NcoI site of the polylinker followedby introduction of the chimeric pTA29-barnase-3′nos gene of pTCO113[WO96/26283] into the EcoRI site of the polylinker. Introduction of thechimeric selectable marker gene pSSUAra-bar-3′g7 in the polylinkersequence of pTPR1 between the T-DNA border repeats results in pTPR3.

[0138] An additional T-DNA vector (pTPR2) is constructed wherein theDZ-selective chimeric gene described above (PDZ2-barnase-3′nos) isaccompanied by the BglII fragment of pTCO113 [WO96/26283] comprising thebarstar coding region under control of the nopaline synthase promoter(pnos-barstar-3′g7) inserted into the polylinker of pTPR1 between theT-DNA border repeats. Introduction of the chimeric selectable markergene pSSUAra-bar-3′g7 in the polylinker sequence of pTPR2 between theT-DNA border repeats results in pTPR4.

EXAMPLE 5

[0139] Construction of a DZ-Selective Chimeric Gene Encoding T-DNA Gene1 Product or the roIB Gene Product.

[0140] A DZ-selective chimeric gene (PDZ-g1-3′nos) is constructedcomprising the following operably linked DNA fragments:

[0141] PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3,comprising a DZ-selective promoter,

[0142] g1: a DNA fragment encoding the Agrobacterium tumefacienstryptophan 2-monooxygenase (iaaM or T-DNA gene 1 product)[Gielen et al.(1984), EMBO J. 3: 835], obtained by polymerase chain reaction usingappropriately desigend primers comprising sequences respectivelyidentical and complementary to the sequences immediately flanking gene1.

[0143]3′nos

[0144] A second DZ-selective chimeric gene (PDZ-g2-3′nos) is constructedcomprising the following operably linked DNA fragments:

[0145] PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3,comprising a DZ-selective promoter,

[0146] g2: a DNA fragment encoding the Agrobacterium tumefaciensindole-3-acetamide hydrolase (iaaH or T-DNA gene 2 product)[Gielen etal. (1984), EMBO J. 3: 835], obtained by polymerase chain reactionamplification, using appropriately desigend primers comprising sequencesrespectively identical and complementary to the sequences immediatelyflanking gene 2.

[0147]3′nos

[0148] Both the DZ-selective chimeric gene (either PDZ-g1-3′nos alone orin combination with PDZ-g2-3′nos) and the PSSU-bar-3′ocs orPSSU-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

[0149] Another DZ-selective chimeric gene (PDZ-roIB-3′nos) isconstructed comprising the following operably linked DNA fragments:

[0150] PDZ: the 5′ regulatory region of Example 3, comprising aDZ-selective promoter, roIB: the open reading frame of the Agrobacteriumrhizogenes roIB gene [Furner et al. (1986), Nature 319: 422]

[0151] 3′nos

[0152] Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs or thePSSU-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

EXAMPLE 6

[0153] Construction of a DZ-Selective Chimeric Gene Encoding a MutantETR1-1 Ethylene Receptor.

[0154] A DZ-selective chimeric gene (PDZ-etr1-1-3′nos) is constructedcomprising the following operably linked DNA fragments:

[0155] PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3,comprising a DZ-selective promoter,

[0156] etr1-1: the open reading frame of the dominant,ethylene-insensitive mutant allele of the Arabidopsis thaliana ETR gene[Chang et al. (1993), Science 262: 539], isolated as 2.7 kb fragmentcomprising the exons of the coding sequence seperated by 5 intronsobtained by PCR amplification using the plasmid carrying the 7.3 kbgenomic EcoRI fragment comprising the DNA of the mutant etr1 allele[Chang et al. (1993), Science 262: 539] and appropriately designedprimers.

[0157] 3′nos

[0158] Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs orPSSUAra-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

EXAMPLE 7

[0159] Construction of a DZ-Selective Chimeric Gene Encoding AntisenseRNA Complementary to mRNA from Which the CDNA of SEQ ID NO: 1 can BePrepared.

[0160] A DZ-selective chimeric gene (PDZ-anti-PG-1-3′nos) is constructedcomprising the following operably linked DNA fragments:

[0161] PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3,comprising a DZ-selective promoter,

[0162] anti-PG-1: a DNA fragment encoding an RNA which is complementaryto the RNA encoded by the region of SEQ ID NO: 1 between nucleotidepositions 10 and 1600.

[0163] 3′nos

[0164] To this end the CaMV35S promoter of the 35S-antisense PGconstruct comprising a DNA sequence complementary to the completesequence of the SEQ ID NO: 1 cloned between a CaMV 35S promoter and apolyadenylation signal (as described below) was eliminated by digestionwith HincII and XhoI, and replaced by the fragment comprising PDZ2.

[0165] Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs orPSSU-bar-3′ocs chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

[0166] Another DZ-selective chimeric gene (PDZ-anti-PG-2-3′nos) isconstructed comprising the following operably linked DNA fragments:

[0167] PDZ or PDZ1 or PDZ2: a 5′ regulatory region of Example 3,comprising a DZ-selective promoter,

[0168] anti-PG-2: a DNA fragment encoding an RNA which is complementaryto the RNA encoded by the region of SEQ ID NO: 1 between nucleotidepositions 20 and 700.

[0169] 3′nos

[0170] Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs or thePSSU-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

[0171] Still another DZ-selective chimeric gene (PDZ-anti-PG-3-3′nos) isconstructed comprising the following operably linked DNA fragments:

[0172] PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3,comprising a DZ-selective promoter,

[0173] anti-PG-3: a DNA fragment encoding an RNA which is complementaryto the RNA encoded by the region of SEQ ID NO: 1 between nucleotidepositions 800 and 1600.

[0174] 3′nos

[0175] Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs or thePSSUAra-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vec tor pGSV5 ofExample 4.

[0176] Three other antisense constructs were constructed comprising aCaMV 35S promoter, a DNA sequence complementary to the complete sequenceof SEQ ID NO: 1, or a DNA sequence complementary to 679 bp of the 3′endof SEQ ID NO: 1 (A67), or a DNA sequence complementary to 336 bp of the3′end of SEQ ID NO: 1 (A30), and a polyadenylation signal. The cDNA ofcDNA-library clone X was excised as a EcoRI-XhoI fragment and insertedin Bluescript® (Stratagene, Calif. USA). The full length cDNA wasisolated as a BamHI-XhoI fragment from this plasmid and inserted intoBamHI-XhoI digested pRT100 vector [Topfer et al (1987) Nucleic AcidResearch 15, 5890], between the CaMV35S promoter and polyadenylationsignal. The resulting plasmid was digested with BamHI and EcoRI, treatedwith Klenow polymerase and self-ligated.

[0177] The HaeIII-XhoI fragment of the Bluescript® plasmid with the cDNAinsert comprising the 3′ end 679 bp of SEQ ID NO: 1 was inserted intothe SmaI-XhoI digested pRT100 vector, between the CaMV35S promoter andpolyadenylation signal, resulting in plasmid A67.

[0178] The A67 construct was digested with Xbal and StyI, treated withKlenow polymerase and self-ligated, resulting in plasmid A30, comprisingthe DNA sequence complementary to 336 bp of the 3′ end of SEQ ID NO: 1between the CaMV35S promoter and polyadenylation signal. The chimericgenes were isolated as PstI fragments.

[0179] 35S-antisense-PG chimeric genes and the PSSU-bar-3′ocs or thePSSU-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector PGSV5 ofExample 4.

EXAMPLE 8

[0180] Transformation of Oilseed Rape and Characterization of theTransformants.

[0181] Agrobacterium-Mediated Transformation.

[0182] Hypocotyl explants of Brassica napus are obtained, cultured andtransformed essentially as described by De Block et al. [(1989), PlantPhysiol. 91: 694), except for the following modifications:

[0183] hypocotyl explants are precultured for 3 days in A2 medium [MS,0.5 g/l Mes (pH5.7), 1.2% glucose, 0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/lnaphthalene acetic acid (NAA)and 1 mg/l 6-benzylaminopurine (BAP)].

[0184] infection medium A3 is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 0.1mg/l NAA, 0.75 mg/l BAP and 0.01 mg/l gibberellinic acid (GA3).

[0185] selection medium A5 is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 40mg/l adenine.SO₄, 0.5 g/l polyvinylpyrrolidone (PVP), 0.5% agarose, 0.1mg/ NAA, 0.75 mg/l BAP, 0.01 mg/l GA3, 250 mg/l carbenicillin, 250 mg/ltriacillin, 0.5 mg/l AgNO₃.

[0186] regeneration medium A6 is MS, 0.5 g/l Mes (pH5.7), 2% sucrose, 40mg/l adenine.SO₄, 0.5 g/l PVP, 0.5% agarose, 0.0025 mg/l BAP and 250mg/l triacillin.

[0187] healthy shoots are transferred to rooting medium which was A8:100-130 ml half concentrated MS, 1% sucrose (pH5.0), 1 mg/l isobutyricacid (IBA), 100 mg/l triacillin added to 300 ml perlite (final pH6.2) in1 liter vessels.

[0188] MS stands for Murashige and Skoog medium [Murashige and Skoog(1962), Physiol. Plant. 15: 473).

[0189] Hypocotyl explants are infected with Agrobacterium tumefaciensstrain C58C1Rif^(R) carrying:

[0190] a helper Ti-plasmid pMP90 [Koncz and Schell (1986), Mol. Gen.Genet 204: 383) or a derivative thereof (such as pGV4000), which isobtained by insertion of a bacterial chloramphenicol resistance genelinked to a 2.5 kb fragment having homology with the T-DNA vector pGSV5,into pMP90.

[0191] T-DNA vector pGSV5 containing between the T-DNA borders theDZ-selective chimeric gene of Example 3, 4, 5, 6, or 7 and the chimericmarker gene.

[0192] Selected lines from these transformants harboring one type of thechimeric genes of the invention are further used for crossingexperiments, yielding new lines comprising combinations of the chimericgenes of the invention.

[0193] Characterization of Transformants.

[0194] Transformed Brassica napus plants of Example 8, comprising intheir nuclear genomes the DZ-selective chimeric gene of Example 3, arecharacterized with respect to the presence of beta-glucuronidase (GUS)activity in various tissues of the plants using conventional in-situhistochemical techniques [De Block and Debrouwer (1992), The PlantJournal 2:261; De Block and Debrouwer (1993), Planta 189: 218]. GUSactivity is only found in the tissues of the pod DZ, attesting to thefact that the DZ-selective promoter of Example 3 directs expressionselectively in the pod DZ.

[0195] Transformed Brassica napus plants of Example 8, comprising intheir nuclear genomes the DZ-selective chimeric genes of either Example4, 5, 6 or 7 alone or in combination are characterized with respect tothe following characteristics:

[0196] 1) changes in physiological processes by analysing diminution ofexpression of targetted gene products (such as cell wall hydrolases) ordiminution in the biochemical activities (see Blumenkratz, supra), bymonitoring the heterologous gene expression (see, Sambrook et al supra),or by measuring the endogenous levels of IAA and IAA conjugates duringdevelopment (see, Example 1)

[0197] 2) changes in DZ anatomy and DZ cell walls during pod senescenceby light microscopy and transmission electron microscopy; the extent ofcell seperation after pod opening by analysing seperated DZ surfaceswith the scanning electron microscope (See, Example 1)

[0198] 3) changes in the mechanical properties of the DZ and their seedshatter resistance by analysing the shatter resistance of individualpods. This can be done by the cantilever test as described by Kadkol etal. [(1986), Aust. J. Bot. 34: 595]. Clamped pods are loaded as acantilever in a “universal testing machine”, consisting of a cross-headbeam moved by actuators to which a load cell applies a constant force todeflect the pod. This records the displacement and the force necessaryto initiate and propagate an opening in the pod dehiscence zone.Alternatively, a first assessment of shatter susceptibility is carriedout with detached pods subjected to controlled vibration (simulatingimpact with canopy and machinery). The vibration consists of horizontaloscillation of fixed amplitute in a container with steel balls toenhance energy transfer. In yet another procedure, susceptibility tocrack propagation is determined by friction measurement. In this case,the force generated due to friction between a wedge forced along the DZis recorded and enables comparison of DZ tissues in selected, extremeexamples of resistance.

[0199] Finally, individual selected lines are subjected to per seperformance analysis in the field. The design of these field trials isbased on the cultivation of individual lines (homozygous for thetransgene) at two locations in three replicates.

[0200] Analysis of a statistically significant number of pods fromdifferent transformed plants demonstrates an increase in pod shatterresistance when compared to untransformed control plants.

[0201] Needless to say, the use of the DZ-selective promoter andrecombinant DNA constructs of this invention is not limited to thetransformation of the specific plant of the examples. Such promoter andrecombinant DNA constructs can be useful in transforming any crop, wherethe promoter can drive gene expression, preferably where such expressionis to occur abundantly in the plant cells of the dehiscence zone.

[0202] Also, the use of the DZ-selective promoter of the presentinvention is not limited to the control of particular transcribed DNAregions of the invention, but can be used to control expression of anyforeign gene or DNA fragment in a plant.

[0203] Furthermore, the present invention is not limited to the specificDZ-selective promoter described in the above Examples. Rather, thepresent invention encompasses promoters, equivalent to the one of theExamples, which can be used to control the expression of a structuralgene, at least substantially selectively in the plant cells of thedehiscence zone. Indeed, the DNA sequence of the DZ-selective promoterof the Examples can be modified by replacing some of its nucleotideswith other nucleotides and/or deleting or inserting some nucleotides,provided that such modifications do not alter substantially the timing,level and tissue-specificity of expression controlled by the promoter,as measured by GUS assays in transgenic plants transformed with achimeric qus gene under control of the modified promoter (see, Example3). Up to 20% of the nucleotides of a promoter may be changed withoutaffecting the characteristics of the promoter. Such promoters can beisolated by hybridization under standard conditions (Sambrook et al,supra) using selected DNA fragments of SEQ ID NO: 14, as describedabove.

[0204] All publications (including patent publications) cited in thisapplication are hereby incorporated by reference.

1 14 1 1631 DNA Brassica napus Location 95-163 = region encoding thepresumed endo-PG signal peptide. 1 ggcacgagaa aaactgcaaa gagtctcatattagttctta ctctcaagaa tcaaacacac 60 tctttctaaa aagattagcg tttcaaaccccgaa atg gcc cgt tgt ttt gga agt 115 Met Ala Arg Cys Phe Gly Ser 1 5 ctagct gtt ttc tta tgc gtt ctt ttg atg ctc gct tgc tgc caa gct 163 Leu AlaVal Phe Leu Cys Val Leu Leu Met Leu Ala Cys Cys Gln Ala 10 15 20 ttg agtagc aac gta gat gat gga tat ggt cat gaa gat gga agc ttc 211 Leu Ser SerAsn Val Asp Asp Gly Tyr Gly His Glu Asp Gly Ser Phe 25 30 35 gaa tcc gatagt tta atc aag ctc aac aac gac gac gac gtt ctt acc 259 Glu Ser Asp SerLeu Ile Lys Leu Asn Asn Asp Asp Asp Val Leu Thr 40 45 50 55 ttg aaa agctct gat aga ccc act acc gaa tca tca act gtt agt gtt 307 Leu Lys Ser SerAsp Arg Pro Thr Thr Glu Ser Ser Thr Val Ser Val 60 65 70 tcg aac ttc ggagcc aaa gga gat gga aaa acc gat gat act cag gct 355 Ser Asn Phe Gly AlaLys Gly Asp Gly Lys Thr Asp Asp Thr Gln Ala 75 80 85 ttc aag aaa gca tggaag aag gca tgt tca aca aat gga gtt act act 403 Phe Lys Lys Ala Trp LysLys Ala Cys Ser Thr Asn Gly Val Thr Thr 90 95 100 ttc tta att cct aaagga aag act tat ctc ctt aag tct att aga ttc 451 Phe Leu Ile Pro Lys GlyLys Thr Tyr Leu Leu Lys Ser Ile Arg Phe 105 110 115 aga ggc cca tgc aaatct tta cgt agc ttc cag atc cta ggc act tta 499 Arg Gly Pro Cys Lys SerLeu Arg Ser Phe Gln Ile Leu Gly Thr Leu 120 125 130 135 tca gct tct acaaaa cga tcg gat tac agt aat gac aag aac cac tgg 547 Ser Ala Ser Thr LysArg Ser Asp Tyr Ser Asn Asp Lys Asn His Trp 140 145 150 ctt att ttg gaagac gtt aat aat cta tca atc gat ggc ggc tcg gcg 595 Leu Ile Leu Glu AspVal Asn Asn Leu Ser Ile Asp Gly Gly Ser Ala 155 160 165 ggg att gtt gatggc aac gga aat atc tgg tgg caa aac tca tgc aaa 643 Gly Ile Val Asp GlyAsn Gly Asn Ile Trp Trp Gln Asn Ser Cys Lys 170 175 180 atc gac aaa tctaag cca tgc aca aaa gcg cca acg gct ctt act ctc 691 Ile Asp Lys Ser LysPro Cys Thr Lys Ala Pro Thr Ala Leu Thr Leu 185 190 195 tac aac cta aagaat ttg aat gtg aag aat ctg aga gtg aga aat gca 739 Tyr Asn Leu Lys AsnLeu Asn Val Lys Asn Leu Arg Val Arg Asn Ala 200 205 210 215 cag cag attcag att tcg att gag aaa tgc aac aat gtt ggc gtt aag 787 Gln Gln Ile GlnIle Ser Ile Glu Lys Cys Asn Asn Val Gly Val Lys 220 225 230 aat gtt aagatc act gct cct ggc gat agt ccc aac acg gat ggt att 835 Asn Val Lys IleThr Ala Pro Gly Asp Ser Pro Asn Thr Asp Gly Ile 235 240 245 cat atc gttgct act aaa aac att cga atc tcc aat tca gac att ggg 883 His Ile Val AlaThr Lys Asn Ile Arg Ile Ser Asn Ser Asp Ile Gly 250 255 260 aca ggt gatgat tgt ata tcc att gag gat gga tcg caa aat gtt caa 931 Thr Gly Asp AspCys Ile Ser Ile Glu Asp Gly Ser Gln Asn Val Gln 265 270 275 atc aat gattta act tgc ggc ccc ggt cat ggg atc agc att gga agc 979 Ile Asn Asp LeuThr Cys Gly Pro Gly His Gly Ile Ser Ile Gly Ser 280 285 290 295 ttg ggggat gac aat tcc aaa gct tat gta tcg gga att gat gtg gat 1027 Leu Gly AspAsp Asn Ser Lys Ala Tyr Val Ser Gly Ile Asp Val Asp 300 305 310 ggt gctacg ctc tct gag act gac aat gga gta aga atc aag act tac 1075 Gly Ala ThrLeu Ser Glu Thr Asp Asn Gly Val Arg Ile Lys Thr Tyr 315 320 325 cag ggaggg tca gga act gct aag aac att aaa ttc caa aac att cgt 1123 Gln Gly GlySer Gly Thr Ala Lys Asn Ile Lys Phe Gln Asn Ile Arg 330 335 340 atg gataat gtc aag aat ccg atc ata atc gac cag aac tac tgc gac 1171 Met Asp AsnVal Lys Asn Pro Ile Ile Ile Asp Gln Asn Tyr Cys Asp 345 350 355 aag gacaaa tgc gaa cag caa gaa tct gcg gtt caa gtg aac aat gtc 1219 Lys Asp LysCys Glu Gln Gln Glu Ser Ala Val Gln Val Asn Asn Val 360 365 370 375 gtgtat cag aac ata aaa ggt acg agc gca aca gat gtg gcg ata atg 1267 Val TyrGln Asn Ile Lys Gly Thr Ser Ala Thr Asp Val Ala Ile Met 380 385 390 tttaat tgc agt gtg aaa tat cca tgc caa ggt att gtg ctt gag aat 1315 Phe AsnCys Ser Val Lys Tyr Pro Cys Gln Gly Ile Val Leu Glu Asn 395 400 405 gtgaac atc aaa gga gga aaa gct tct tgc gaa aat gtc aat gtt aag 1363 Val AsnIle Lys Gly Gly Lys Ala Ser Cys Glu Asn Val Asn Val Lys 410 415 420 gataaa ggc act gtt tct cct aaa tgc cct taattactaa gctgattatg 1413 Asp LysGly Thr Val Ser Pro Lys Cys Pro 425 430 taatatacat aaatacgtag tatatntaattatagatgca tgtatatcgt tatctacgta 1473 ttgattcttg atatatatag aaaactaaagatatatggga atatacatac aatagttgag 1533 ataattgttg tcttgtatat gattcactgaagttgattgc ttgtccatga ataaatgaat 1593 aatatcattt ctctaaaaaa aaaaaaaaaaaaaaaaaa 1631 2 433 PRT Brassica napus Strain cv. Topaz. 2 Met Ala ArgCys Phe Gly Ser Leu Ala Val Phe Leu Cys Val Leu Leu 1 5 10 15 Met LeuAla Cys Cys Gln Ala Leu Ser Ser Asn Val Asp Asp Gly Tyr 20 25 30 Gly HisGlu Asp Gly Ser Phe Glu Ser Asp Ser Leu Ile Lys Leu Asn 35 40 45 Asn AspAsp Asp Val Leu Thr Leu Lys Ser Ser Asp Arg Pro Thr Thr 50 55 60 Glu SerSer Thr Val Ser Val Ser Asn Phe Gly Ala Lys Gly Asp Gly 65 70 75 80 LysThr Asp Asp Thr Gln Ala Phe Lys Lys Ala Trp Lys Lys Ala Cys 85 90 95 SerThr Asn Gly Val Thr Thr Phe Leu Ile Pro Lys Gly Lys Thr Tyr 100 105 110Leu Leu Lys Ser Ile Arg Phe Arg Gly Pro Cys Lys Ser Leu Arg Ser 115 120125 Phe Gln Ile Leu Gly Thr Leu Ser Ala Ser Thr Lys Arg Ser Asp Tyr 130135 140 Ser Asn Asp Lys Asn His Trp Leu Ile Leu Glu Asp Val Asn Asn Leu145 150 155 160 Ser Ile Asp Gly Gly Ser Ala Gly Ile Val Asp Gly Asn GlyAsn Ile 165 170 175 Trp Trp Gln Asn Ser Cys Lys Ile Asp Lys Ser Lys ProCys Thr Lys 180 185 190 Ala Pro Thr Ala Leu Thr Leu Tyr Asn Leu Lys AsnLeu Asn Val Lys 195 200 205 Asn Leu Arg Val Arg Asn Ala Gln Gln Ile GlnIle Ser Ile Glu Lys 210 215 220 Cys Asn Asn Val Gly Val Lys Asn Val LysIle Thr Ala Pro Gly Asp 225 230 235 240 Ser Pro Asn Thr Asp Gly Ile HisIle Val Ala Thr Lys Asn Ile Arg 245 250 255 Ile Ser Asn Ser Asp Ile GlyThr Gly Asp Asp Cys Ile Ser Ile Glu 260 265 270 Asp Gly Ser Gln Asn ValGln Ile Asn Asp Leu Thr Cys Gly Pro Gly 275 280 285 His Gly Ile Ser IleGly Ser Leu Gly Asp Asp Asn Ser Lys Ala Tyr 290 295 300 Val Ser Gly IleAsp Val Asp Gly Ala Thr Leu Ser Glu Thr Asp Asn 305 310 315 320 Gly ValArg Ile Lys Thr Tyr Gln Gly Gly Ser Gly Thr Ala Lys Asn 325 330 335 IleLys Phe Gln Asn Ile Arg Met Asp Asn Val Lys Asn Pro Ile Ile 340 345 350Ile Asp Gln Asn Tyr Cys Asp Lys Asp Lys Cys Glu Gln Gln Glu Ser 355 360365 Ala Val Gln Val Asn Asn Val Val Tyr Gln Asn Ile Lys Gly Thr Ser 370375 380 Ala Thr Asp Val Ala Ile Met Phe Asn Cys Ser Val Lys Tyr Pro Cys385 390 395 400 Gln Gly Ile Val Leu Glu Asn Val Asn Ile Lys Gly Gly LysAla Ser 405 410 415 Cys Glu Asn Val Asn Val Lys Asp Lys Gly Thr Val SerPro Lys Cys 420 425 430 Pro 3 27 DNA Artificial Sequence Description ofArtificial Sequenceoligo- nucleotide PG1 3 ccaggaattc aayacngayg gnrtnca27 4 25 DNA Artificial Sequence Description of Artificial Sequenceoligo-nucleotide PG2 4 cgacggatcc angtyttdat nckna 25 5 27 DNA ArtificialSequence Description of Artificial Sequenceoligo- nucleotide PG3 5ggacgaattc acnggngayg aytgyat 27 6 27 DNA Artificial SequenceDescription of Artificial Sequenceoligo- nucleotide PG5 6 cacaggatccswngtnccny kdatrtt 27 7 155 DNA Brassica napus unsure (13)..(19) PCRfragment BPG32-26 from first strand cDNA. Strain cv. Topaz 7 tcgattcaaaccggttgctc caatgtgtat gttcacaatg tgaattgtgg accaggacat 60 ggcatcagcatagggagtct tggtaaagac agtaccaaag cttgtgtctc caatataaca 120 gtcagagatgtagttatgca caacacaatg actgg 155 8 155 DNA Brassica napus PCR fragmentKPG32-8 from first strand cDNA. Strain cv. Topaz. 8 tctattggagacgggacgag agaccttctt gtcgaaagag ttacatgcgg tccgggacat 60 ggaatcagtattggaagcct cggtttatac gtgaaggagg aagacgtcac tggcatcagg 120 gtcgtgaactgcaccctcat aaacactgac aatgg 155 9 219 DNA Brassica napus PCR fragmentLPG12-16 from first strand cDNA. Strain cv. Topaz. 9 tttgggaagaagtgacggag tcaagatcct taacacattc atctccaccg gagacgactg 60 tatctccgttggagatggga tgaagaacct tcacgtggag aaagtcacct gcggtccagg 120 acatggaatcagtgtcggaa gccttggaag gtacggaaac gaacaggatg tcagcggcat 180 tagagtcataaactgcactc tccaacagac tgacaacgg 219 10 155 DNA Brassica napus PCRfragment LPG32-24 from first strand cDNA. Strain cv. Topaz. 10tccattggag gcggtactga aaatttactt gtcgagggcg tagaatgtgg accaggacac 60ggtctttcca tcggaagtct tggaaagtac cctaatgagc aaccagtgaa aggaatcacc 120attcgtaaat gcatcatcaa gcataccgat aatgg 155 11 155 DNA Brassica napus PCRfragment LPG32-25 from first strand cDNA. Strain cv. Topaz. 11tctgttgggg acgggatgaa aaaccttctt gtcgaaagag tttcatgcgg tccgggacac 60ggaatcagta ttggaagcct cggattatac gggcacgagg aagacgtcac tggcgtcaag 120gtcgtgaact gcaccctcag aaatactgac aatgg 155 12 155 DNA Brassica napus PCRfragment LPG32-32 from first strand cDNA. Strain cv. Topaz. 12tccgtgggag atgggatgaa gaatctcctc attgagaaag ttgtgtgcgg tccaggacac 60ggaatcagtg ttggaagcct tggaaggtac ggatgggagc aagatgtcac tgacattaac 120gttaagaact gtaccctcga gggaaccgac aacgg 155 13 100 DNA ArtificialSequence Description of Artificial SequenceDNA sequence of the T-DNA ofpGSV5 13 aattacaacg gtatatatcc tgccagtact cggccgtcga ccgcggtacccggggaagct 60 tagatccatg gagccattta caattgaata tatcctgccg 100 14 2352DNA Brassica napus Location 2329-2331 = translation initiation codon. 14acgagaatcg agagaaacaa aaactctcgt cgcaagcaca agtttggggt aggttgtatg 60gtgtaagaat gacgggccat agagaataat gtcttccact cttgccaaac ggactgaaac 120catcagtaca taatccaagg tagacatttc ttctctcata cgcaaagtcg ggatactttg 180attggaaatg cttccacgct tttgcatctg aaggatgtct gatctcacca tctgttgagt 240gctccgcatg ccatctcatt ggttgcgctg tgcgttcaga cagatacaac ctctgcaacc 300tttccgtcaa aggtaaatac cacatccttt tatatggcac tggaactctt ccactcgtat 360ctttataacg aggctttcca caaaatttgc atgtaacccg ctgttcatcc gccctccaat 420aaatcatgca gttgtcgctg catacatcta ttacctgata cgccaagacc agctacgagt 480ttctgaacct cgtagtatga accaggagct acattattct cgggtagaat accttttaca 540aaatcagcaa tcgcatccac acagtcttca gccaaattat aatctttttc aatgcccatc 600aatcttgtag cagatgataa agctgaatga ccatctctgc aaccttcgta caatggttgc 660tttccagcat ccaacatatc ataaaatttc ctagcttgtg cattgggtaa atcttcccct 720ctaaaatgat catttaccat ctgctcagta cctacaccat aatctacatc cgttctaatt 780ggttcttcta atctaaccgc tggctgaggt tcgctagtac taccatgttc ataatcagtt 840tccccatgat gataccaaat tttgtaactt cgtgtaaacc cactcaaata tagatgagtc 900caaacatccc actctttaat aacttttcta tttttacaat tagagcaaga acatcttaac 960atacatgttt ttgcttccgg ttgtcggtga actaacccca tgaattcggt tatacctcgt 1020tggtattctt ccgtaagcaa tctcgtgttc ggatccaaat gaggtcgatc gatccaagaa 1080cgaaaataat ttgaagaaga catatttttt atgaatcaaa ttcgtgtgta aatagagtaa 1140gagggaggat gaagatatgg agtgaatgaa gaggaagagg agtgcttgta tttatagttt 1200aaatcctgcc gacagaccga ggaaattccg acggaattcc gacggaaaag gctagttcgt 1260cggaatttcc tcggaatttt gtaaaatccc ccagcggctc tccaacggct ataatatttc 1320ctcggaattc atcggttttt tccgaggaac acatttttcc tcggaatttc ctcggaatat 1380tccaacggat tgatatttcc tcggaattcc gtcggtatat tccaaggaaa cccaattttg 1440tgtttcctca gaatttcctc agaaattcct cgggatattc cgaggatttc attttccgtc 1500ggaatgtcca tcagaatacc gctgttttct tgtagtgatt attatttttt ttttcagata 1560aaaaaaaaag aaatatcaac caatcgctga ctgtcacata ttgtgggggc ccacaaatag 1620tgcaaggact cactaagaaa aagtttttat tttagaattt tagtaattga attcttaact 1680tttggtggag ttcactgatt atttaattat ttttttttaa gaactcaccc ttaagaattg 1740ttgttgcgga tgttctaata tcagcatcac acaccaaaat aaaaagcacg aaagagtaaa 1800agggacccaa cactactatc gaactttgaa agacggttga cgccgacgtt tatcactttt 1860gcttatatgt tttcaacttt ttatatctaa tgtagggata tatacatcac gtaatgttag 1920ctcagtaatt gcacatgatg gaatgttact gtgaatggta tacgatgatg aatataaact 1980cttttctagt agaaaataac taactaatta aactctctat caatcaagaa agcaataaaa 2040atcaataaaa agataaatta aaatggaggg gagaggagat aaaggttaga agctagggtg 2100tgatgttttc gtatcaatct caatctctct ccatacctcc aacgccatta atacttgaat 2160aaacatataa aatttctcca ttgaattgcc tataaataca catacatccc acttcttcaa 2220tttcatatta caaaagcctc ccaaaaactg caaagagtct catattagtt cttactctca 2280agaatcaaac acactctttc taaaaagatt agcgtttcaa accccgaaat ggcccgttgt 2340tttggaagtc ta 2352

1. An isolated DNA fragment comprising a nucleotide sequence encoding anendo-polygalacturonase, said nucleotide sequence having at least 90%sequence identity to SEQ ID NO:
 1. 2. The DNA fragment according toclaim 1, wherein said DNA fragment is obtained from Brassica napuscultivar Topaz or cultivar Bridger.
 3. An isolated DNA fragmentcomprising a nucleotide sequence encoding an endo-polygalacturonase,said DNA fragment hybridizing under high stringency hybridizationconditions with a DNA fragment having the nucleotide sequence of SEQ IDNO:
 1. 4. An isolated DNA fragment comprising the nucleotide sequence ofSEQ ID NO:1 from the nucleotide at position 10 to the nucleotide atposition
 1600. 5. An isolated DNA fragment comprising a nucleotidesequence encoding a signal peptide of an endopolygalacturonasecomprising the amino acid sequence of SEQ ID NO:2 from the amino acid atposition 1 to the amino acid at position
 24. 6. An isolated DNA fragmentcomprising a nucleotide sequence encoding an endopolygalacturonasecomprising the amino acid sequence of SEQ ID NO:2 from the amino acid atposition 24 to the amino acid at position
 433. 7. An isolated DNAfragment comprising a nucleotide sequence encoding anendopolygalacturonase comprising the amino acid sequence of SEQ ID NO:2from the amino acid at position 1 to the amino acid at position
 433. 8.A vector comprising the DNA fragment according to any one of claims 1 to7.
 9. A chimeric gene comprising the following operationally linkedelements: (a) a plant expressible promoter, which is at least active inthe pod dehiscence zone of a Brassica plant; and (b) a DNA fragment,which is transcribed into an antisense RNA, which, when produced incells of a pod dehiscence zone of said Brassica plant, prevents,inhibits or reduces in said cells the expression of an endogenous geneencoding an endo-polygalacturonase, wherein said RNA is complementary topart or all of the RNA transcribed from the 5′ untranslated region, thecoding region, an intron or the 3′ untranslated region of a Brassicaendopolygalacturonase gene, said endopolygalacturonase gene hybridizingunder high stringency hybridization conditions with a DNA fragmenthaving the nucleotide sequence of SEQ ID NO:
 1. 10. The chimeric geneaccording to claim 9, wherein said Brassica endo-polygalacturonase genecomprises the nucleotide sequence of SEQ ID NO:1.
 11. The chimeric geneaccording to claim 10, wherein said antisense RNA comprises a continuousstretch of at least 50 nucleotides.
 12. The chimeric gene according toclaim 10, wherein said antisense RNA comprises a continuous stretch ofat least 100 nucleotides.
 13. The chimeric gene according to claim 10,wherein said antisense RNA comprises a continuous stretch of at least1000 nucleotides.
 14. A chimeric gene comprising the followingoperationally linked elements: (a) a plant expressible promoter, whichis at least active in the pod dehiscence zone of a Brassica plant, (b)and a DNA fragment, encoding a sense RNA, which, when produced in cellsof a pod dehiscence zone of a Brassica plant, prevents, inhibits orreduces in said cells the expression of an endogenous gene encoding anendopolygalacturonase, wherein said RNA is identical to part or all ofthe RNA encoded by the 5′ untranslated region, the coding region, anintron or the 3′ untranslated region of a Brassicaendo-polygalacturonase gene, said endopolygalacturonase gene hybridizingunder high stringency hybridization conditions with a DNA fragmenthaving the nucleotide sequence of SEQ ID NO:
 1. 15. The chimeric geneaccording to claim 9 or 14, wherein the promoter is a constitutivepromoter or a dehiscence zone specific promoter.
 16. A Brassica plantwith modified pod-dehiscence properties, comprising a chimeric geneaccording to claim 9 or 14 incorporated in its nuclear genome.
 17. ABrassica plant cell or plant cell culture comprising a chimeric geneaccording to claim 9 or 14 incorporated in its nuclear genome.
 18. ABrassica seed comprising a chimeric gene according to claim 9 or 14incorporated in its nuclear genome.
 19. A method for producing aBrassica plant with modified pod dehiscence properties, comprising thesteps of: (a) transforming the nuclear genome of a Brassica plant cellwith a chimeric gene according to claim 9 or 14, and (b) regenerating atransformed Brassica plant from said transformed plant cell.